Systems and devices for analysis of samples

ABSTRACT

Systems and methods for analysis of samples, and in certain embodiments, microfluidic sample analyzers configured to receive a cassette containing a sample therein to perform an analysis of the sample are described. The microfluidic sample analyzers may be used to control fluid flow, mixing, and sample analysis in a variety of microfluidic systems such as microfluidic point-of-care diagnostic platforms. Advantageously, the microfluidic sample analyzers may be, in some embodiments, inexpensive, reduced in size compared to conventional bench top systems, and simple to use. Cassettes that can operate with the sample analyzers are also described.

RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 13/088,102, filed Apr. 15, 2011 and entitled “Systems and Devicesfor Analysis of Samples,” which claims priority under 35 U.S.C. §119(e)to U.S. provisional applications: U.S. Ser. No. 61/325,023, filed Apr.16, 2010 and entitled “Feedback Control in Microfluidic Systems”, U.S.Ser. No. 61/325,044, filed Apr. 16, 2010 and entitled “Feedback Controlin Microfluidic Systems”, and U.S. Ser. No. 61/363,002, filed Jul. 9,2010 and entitled “Systems and Devices for Analysis of Samples”, each ofwhich is incorporated herein by reference in its entirety.

FIELD

The present application relates generally to systems, devices andmethods for analysis of samples, and in certain embodiments, tomicrofluidic sample analyzers configured to receive a cassette having asample therein to analyze the sample. Cassettes for sample analysis arealso provided.

BACKGROUND

The manipulation of fluids plays an important role in fields such aschemistry, microbiology and biochemistry. These fluids may includeliquids or gases and may provide reagents, solvents, reactants, orrinses to chemical or biological processes. While various microfluidicmethods and cassettes, such as microfluidic assays, can provideinexpensive, sensitive and accurate analytical platforms, fluidmanipulations—such as sample introduction, introduction of reagents,storage of reagents, control of fluid flow, separation of fluids, mixingof multiple fluids, collection of waste, extraction of fluids foroff-chip analysis, and/or transfer of fluids from one chip to thenext—can add a level of cost and sophistication. Often, a microfluidiccassette requires an external platform such as an analyzer to performsome such and other fluid manipulations. Various types of analyzersexist to process and analyze a microfluidic sample, however, some suchanalyzers are expensive, bulky, difficult to use, and/or require complexcomponents for manipulating fluids. Accordingly, advances in the fieldthat could reduce costs, reduce size, simplify use, reduce complexity ofcomponents required for fluid manipulations, and/or improve fluidmanipulations in microfluidic systems would be beneficial.

SUMMARY

Systems and methods for analysis of samples are described. The subjectmatter of the present invention involves, in some cases, interrelatedproducts, alternative solutions to a particular problem, and/or aplurality of different uses of one or more systems and/or articles.

In one set of embodiments, a series of methods are provided. In oneembodiment, a method of analyzing a microfluidic sample comprises thesteps of providing a microfluidic sample analyzer comprising a housingwith an opening therein, wherein a cassette is contained in the openingin the housing and, wherein the cassette or a component of the cassetteincludes at least one channel with a fluid sample therein. The methodincludes identifying information about the cassette with anidentification reader positioned within the housing, and processinginformation input by a user into a user interface positioned within thehousing of the sample analyzer. The method also involves pressurizingthe at least one channel in the cassette with a pressure-control systempositioned within the housing to move the sample through the at leastone channel. The method includes activating an optical system thatpasses light from a first light source positioning within the housingthrough a first measurement zone of the cassette, and detecting theamount of light transmission through the first measurement zone of thecassette with a first detector of the optical system positioned withinthe housing opposite the first light source. The method involvesanalyzing the sample in the cassette with a control system positionedwithin the housing which communicates with the identification reader,the user interface, the pressure-control system, the optical system andthe temperature regulating system. The method may optionally includeheating the cassette with a temperature regulating system positionedwithin the housing of the sample analyzer.

In another set of embodiments, a series of microfluidic sample analyzersare provided. In one embodiment, a microfluidic sample analyzercomprises a housing, an opening in the housing configured to receive acassette having at least one channel with a fluid sample therein,wherein the housing includes a component configured to interface with amating component on the cassette to detect the cassette within thehousing, and an identification reader positioned within the housing andconfigured to read information associated with the cassette. Themicrofluidic sample analyzer also includes a user interface positionedwithin the housing and configured for a user to input information intothe sample analyzer, and a pressure-control system positioned within thehousing, the pressure-control system configured to pressurize the atleast one channel in the cassette to move the sample through the atleast one channel. The microfluidic sample analyzer further includes anoptical system positioned within the housing, the optical systemincluding at least a first light source and a first detector spacedapart from the first light source, wherein the first light source isconfigured to pass light through a first measurement zone of a cassettewhen inserted into the sample analyzer and wherein the first detector ispositioned opposite the first light source to detect the amount of lighttransmission through the first measurement zone of the cassette. Themicrofluidic sample analyzer also includes a temperature regulatingsystem positioned within the housing, the temperature regulating systemincluding a heater configured to heat the cassette, and a control systempositioned within the housing and configured to communicate with theidentification reader, the user interface, the pressure-control system,the optical system and the temperature regulating system, to analyze thesample in the cassette.

In another embodiment, a microfluidic sample analyzer comprises ahousing, and an opening in the housing configured to receive a cassettehaving at least one channel with a fluid sample therein and at least onemicrofluidic channel having a cross-sectional dimension of less than 1mm, wherein the housing includes a component configured to interfacewith a mating component on the cassette to detect the cassette withinthe housing. The microfluidic sample analyzer includes apressure-control system positioned within the housing, thepressure-control system configured to pressurize the at least onechannel in the cassette to move the sample through the at least onechannel, and an optical system positioned within the housing, theoptical system including a plurality of light sources and a plurality ofdetectors spaced apart from the plurality of light sources, wherein thelight sources are configured to pass light through the cassette when thecassette is inserted into the sample analyzer and wherein the detectorsare positioned opposite the light sources to detect the amount of lightthat passes through the cassette. The plurality of light sourcesincludes at least a first light source and a second light sourceadjacent the first light source, wherein the first light source isconfigured to pass light through a first measurement zone of thecassette and the second light source is configured to pass light througha second measurement zone of the cassette adjacent the first measurementzone. In some embodiments, the light sources are configured such thatsecond light source is not activated unless the first light source isdeactivated.

In another embodiment, a microfluidic sample analyzer comprises ahousing, and an opening in the housing configured to receive a cassettehaving at least one channel with a fluid sample therein. Themicrofluidic sample analyzer further includes a spring loaded armpositioned within the housing that has a first position where the armextends at least partially across the opening in the housing such thatas a cassette is inserted into the opening in the housing, the springloaded arm is pushed away from the opening into a second position, andwherein the spring loaded arm is configured to contact a side surface ofthe cassette as the cassette is inserted into the housing until thespring loaded arm engages an inwardly cammed surface in the cassettethat is configured such that the spring loaded arm returns towards thefirst position where it extends at least partially across the opening inthe housing to retain the cassette within the housing.

In another embodiment, a series of microfluidic sample cassettes areprovided. In one embodiment, a microfluidic sample cassette comprises ahousing comprising at least one microfluidic channel extending throughat least a portion of the housing, wherein the channel is configured toreceive a sample therein. The housing comprises at least one surfaceconfigured to interact with a microfluidic sample analyzer such that thesample cassette may be inserted into and retained within the sampleanalyzer, the at least one surface of the housing comprising a curvedcorner surface configured to contact an arm within the sample analyzer,and a notch configured to retain the arm of the sample analyzer.

In one set of embodiments, a kit is provided. The kit includes a firstcomponent comprising a first channel in a first material, the firstchannel including an inlet, an outlet and, between the first channelinlet and outlet, at least one portion having a cross-sectionaldimension greater than 200 microns. The kit also includes a secondcomponent comprising a second channel in a second material, the secondchannel including an inlet, an outlet and, between the second channelinlet and outlet, at least one portion having a cross-sectionaldimension less than 200 microns. In some embodiments, the first materialis different from the second material (although in other embodiments,the first material may be the same as the second material). In someembodiments, the first material has a water vapor permeability of lessthan about 0.05 g·mm/mm²·d. In certain embodiments, the second materialhas an optical transmission of greater than 90% between 400 nm and 800nm wavelengths of light. The kit also includes a fluidic connector forfluidly connecting the first and second channels, the fluidic connectorcomprising a fluid path including a fluid path inlet and a fluid pathoutlet. The fluid path inlet can be fluidly connected to the outlet ofthe first channel and the fluid path outlet can be fluidly connected tothe inlet of the second channel. The kit is packaged such that thefluidic connector is not fluidically connecting the first and secondchannels.

In another set of embodiments, a device is provided. The device includesa first component comprising a first channel formed in a first materialand including at least one inlet and one outlet, the first channelincluding at least one portion having a cross-sectional dimensiongreater than 200 microns. The device also includes a second componentcomprising a second channel formed in a second material and including atleast one inlet and one outlet, the second channel including at leastone portion having a cross-sectional dimension less than 200 microns. Insome embodiments, the first material is different from the secondmaterial (although in other embodiments, the first material may be thesame as the second material). In some embodiments, the first materialhas a water vapor permeability of less than about 0.05 g·mm/mm²·d. Incertain embodiments, the second material has an optical transmission ofgreater than 90% between 400 nm and 800 nm wavelengths of light. Thedevice also includes a fluidic connector that can be connected to thefirst and second components, the fluid connector comprising a fluid pathincluding a fluid path inlet and a fluid path outlet, wherein uponconnection, the fluid path inlet fluidically connects to the outlet ofthe first channel and the fluid path outlet fluidically connects to theinlet of the second channel to allow fluid communication between thefirst and second channel. The first and second channels are not in fluidcommunication with one another prior to first use, and at first use, thefirst and second channels are brought into fluid communication with oneanother.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is typically represented by a likedescriptor. For purposes of clarity, not every component may be labeledin every drawing.

Various embodiments will now be described, by way of example, withreference to the accompanying drawings, in which:

FIG. 1A is a block diagram showing a microfluidic system and a varietyof components that may be part of a sample analyzer according to oneembodiment;

FIG. 1B is a perspective view of a sample analyzer and cassetteaccording to one embodiment;

FIG. 2 is a perspective view of the internal components of a sampleanalyzer according to one embodiment with the housing removed;

FIG. 3 is a perspective view of a cassette including a fluidic connectoraccording to one embodiment;

FIG. 4 is a perspective view showing the insertion of a fluidicconnector into a portion of a cassette according to one embodiment;

FIG. 5 is an exploded assembly view of a fluidic connector according toone embodiment;

FIG. 6 is a perspective view of a cassette according to one embodiment;

FIG. 7 is a an exploded assembly view of a cassette according to oneembodiment;

FIG. 8 is a schematic view of a cassette including a fluidic connectoraccording to one embodiment;

FIG. 9A is a schematic view of a cassette according to one embodiment;

FIGS. 9B-9F are schematic views of cassettes formed of multiplecomponents according to one set of embodiments;

FIG. 10 is a partial assembly view of a sample analyzer according to oneembodiment;

FIG. 11 is a top view of a partial assembly of a sample analyzeraccording to one embodiment;

FIG. 12 is another top view of a partial assembly of a sample analyzeraccording to one embodiment;

FIG. 13 is a schematic view of a portion of a sample analyzer accordingto one embodiment;

FIG. 14 is a schematic side view of a portion of a sample analyzeraccording to one embodiment;

FIG. 15 is a perspective view of a vacuum system of a sample analyzeraccording to one embodiment;

FIG. 16 is a block diagram showing a control system of a sample analyzerassociated with a variety of different components according to oneembodiment;

FIGS. 17-21 are schematic views of a user interface of a sample analyzeraccording to one embodiment;

FIG. 22 is a schematic diagram showing a microfluidic system of acassette according to one embodiment; and

FIG. 23 is a plot showing measurement of optical density as a functionof time according to one embodiment.

DETAILED DESCRIPTION

Systems and methods for analysis of samples, and in certain embodiments,microfluidic sample analyzers configured to receive a cassettecontaining a sample therein to perform an analysis of the sample aredescribed.

Applicant recognized the need for a unique microfluidic sample analyzerwhich may be configured to process a sample to measure the level of oneor more analytes (e.g., a prostate specific antigen (PSA)) in thesample. As set forth below, measuring the PSA level or level of otheranalytes in a blood sample may help manage prostate cancer or otherdisease and/or conditions.

Microfluidic sample analyzers described herein may also be configuredand used to process a sample for other reasons, as the invention is notlimited to a particular application. For example, in one embodiment, themicrofluidic sample analyzers discussed herein may be configured forvarious types of protein analysis and and/or DNA and/or RNA analysis. Insome cases, the systems and methods described herein can be used tocontrol fluid flow and mixing in a variety of microfluidic systems suchas, for example, microfluidic point-of-care diagnostic platforms,microfluidic laboratory chemical analysis systems, fluidic controlsystems in cell cultures or bio-reactors, among others. In oneembodiment, the microfluidic sample analyzer is configured for varioustypes of hematology and/or urology applications. The microfluidic sampleanalyzers discussed herein may be configured for a wide variety ofdiagnostics and general chemical and/or biological analysis. The sampleanalyzer may be specifically configured for a particular applicationand/or may be configured to analyze a sample according to a variety ofthe applications discussed above and herein.

As set forth in more detail below, the microfluidic sample analyzer maybe configured to receive a cassette which includes at least one channelwith a sample contained therein. The sample cassette may be configuredto be a disposable component that is discarded after the sample isanalyzed.

The articles, components, systems, and methods described herein may becombined with those described in International Patent Publication No.WO2005/066613 (International Patent Application Serial No.PCT/US2004/043585), filed Dec. 20, 2004 and entitled “Assay Device andMethod”; International Patent Publication No. WO2005/072858(International Patent Application Serial No. PCT/US2005/003514), filedJan. 26, 2005 and entitled “Fluid Delivery System and Method”;International Patent Publication No. WO2006/113727 (International PatentApplication Serial No. PCT/US06/14583), filed Apr. 19, 2006 and entitled“Fluidic Structures Including Meandering and Wide Channels”; U.S. patentapplication Ser. No. 12/113,503, published as U.S. Patent PublicationNo. 2008/0273918, filed May 1, 2008 and entitled “Fluidic Connectors andMicrofluidic Systems”; U.S. patent application Ser. No. 12/196,392,published as U.S. Patent Publication No. 2009/0075390, filed Aug. 22,2008, entitled “Liquid containment for integrated assays”; U.S. patentapplication Ser. No. 12/428,372, filed Apr. 22, 2009, published as U.S.Patent Publication No. 2009/0266421, entitled “Flow Control inMicrofluidic Systems”; U.S. patent application Ser. No. 12/640,420,published as U.S. Patent Publication No. 2010/0158756, filed Dec. 17,2009, entitled, “Reagent Storage in Microfluidic Systems and RelatedArticles and Methods”; U.S. patent application Ser. No. 12/698,451,filed Feb. 2, 2010, entitled, “Structures for Controlling LightInteraction with Microfluidic Devices”; U.S. Patent Apl. Ser. No.61/263,981, filed Nov. 14, 2009 and entitled, “Fluid Mixing and Deliveryin Microfluidic Systems; U.S. Provisional Patent Application No.61/325,044, filed Apr. 16, 2010 entitled, “System for Analysis ofSamples”; and U.S. Provisional Patent Application No. 61/325,023, filedApr. 16, 2010 entitled, “Feedback Control in Microfluidic Systems”, eachof which is incorporated herein by reference in its entirety for allpurposes.

A series of exemplary systems and methods are now described.

FIG. 1A shows a block diagram 10 of a microfluidic system and variouscomponents that may provide feedback control according to one set ofembodiments. The microfluidic system may include, for example, acassette 20 operatively associated with one or more components such as afluid flow source 40 such as a pump (e.g., for introducing one or morefluids into the cassette and/or for controlling the rates of fluidflow), optionally a fluid flow source 40 such as a pump or vacuum thatmay be configured to apply either of both of a positive pressure orvacuum (e.g., for moving/removing one or more fluids within/from thecassette and/or for controlling the rates of fluid flow), a valvingsystem 28 (e.g., for actuating one or more valves), a detection system34 (e.g., for detecting one or more fluids and/or processes), and/or atemperature regulating system 41 (e.g., to heat and/or cool one or moreregions of the cassette). The components may be external or internal tothe microfluidic device, and may optionally include one or moreprocessors for controlling the component or system of components. Incertain embodiments, one or more such components and/or processors areassociated with a sample analyzer 47 configured to process and/oranalyze a sample contained in the cassette.

In general, as used herein, a component that is “operatively associatedwith” one or more other components indicates that such components aredirectly connected to each other, in direct physical contact with eachother without being connected or attached to each other, or are notdirectly connected to each other or in contact with each other, but aremechanically, electrically (including via electromagnetic signalstransmitted through space), or fluidically interconnected (e.g., viachannels such as tubing) so as to cause or enable the components soassociated to perform their intended functionality.

The components shown illustratively in FIG. 1A, as well as otheroptional components such as those described herein, may be operativelyassociated with a control system 50. In some embodiments, the controlsystem may be used to control fluids and/or conduct quality control bythe use of feedback from one or more events taking place in themicrofluidic system. For instance, the control system may be configuredto receive input signals from the one or more components, to calculateand/or control various parameters, to compare one or more signals or apattern of signals with signals preprogrammed into the control system,and/or to send signals to one or more components to modulate fluid flowand/or control operation of the microfluidic system, as described inmore detail herein and in a U.S. Provisional Patent Application No.61/325,023, filed Apr. 16, 2010 and entitled, “Feedback Control inMicrofluidic Systems”, which is incorporated herein by reference in itsentirety for all purposes. The control system may also be optionallyassociated with other components such as a user interface 54, anidentification system 56, an external communication unit 58 (e.g., aUSB), and/or other components, as described in more detail below.

Cassette (e.g., microfluidic device) 20 may have any suitableconfiguration of channels and/or components for performing a desiredanalysis. In one set of embodiments, cassette 20 contains storedreagents that can be used for performing a chemical and/or biologicalreaction (e.g., an immunoassay), e.g., as described in more detailherein. The cassette may include, for example, an optional reagent inlet62 in fluid communication with an optional reagent storage area 64. Thestorage area may include, for example, one or more channels and/orreservoirs that may, in some embodiments, be partially or completelyfilled with fluids (e.g., liquids and gases, including immisciblereagents such as reagent solutions and wash solutions, optionallyseparated by immiscible fluids, as described in more detail below). Thecassette may also include an optional sample or reagent loading area 66,such as a fluidic connector that can be used to connect reagent storagearea 64 to an optional measurement zone 68. The measurement zone, whichmay include one or more areas for detecting a component in a sample(e.g., measurement zones), may be in fluid communication with anoptional waste area 70 and coupled to outlet 72. In some cases, such andother device features may be formed on or in different components orlayers of a cassette, as described in more detail herein. Thus, itshould be appreciated that a cassette may include a single component, ormultiple components that are attached during use, such as a combinationof an article with attached fluidic connector as described herein. Inone set of embodiments, fluid may flow in the direction of the arrowsshown in the figure. Further description and examples of such and othercomponents are provided in more detail below.

In some embodiments, sections 71 and 77 of the cassette are not in fluidcommunication with one another prior to introduction of a sample intothe cassette. In some cases, sections 71 and 77 are not in fluidcommunication with one another prior to first use of the cassette,wherein at first use, the sections are brought into fluid communicationwith one another. In other embodiments, however, sections 71 and 77 arein fluid communication with one another prior to first use and/or priorto introduction of a sample into the cassette. Other configurations ofcassettes are also possible. As shown in the exemplary embodimentillustrated in FIG. 1A, one or more fluid flow sources 40 such as a pumpand/or a vacuum or other pressure-control system, valving system 28,detection system 34, temperature regulating system 41, and/or othercomponents may be operatively associated with one or more of reagentinlet 62, reagent storage area 64, sample or reagent loading area 66,reaction area 68, waste area 70, outlet 72, and/or other regions ofcassette 20. Detection of processes or events in one or more regions ofthe cassette can produce a signal or pattern of signals that can betransmitted to control system 50. Based on the signal(s) received by thecontrol system, this feedback can be used to manipulate fluids withinand/or between each of these regions of the microfluidic device, such asby controlling one or more of a pump, vacuum, valving system, detectionsystem, temperature regulating system, and/or other components.

Turning to FIGS. 1B-2, one embodiment of a microfluidic sample analyzer100 is illustrated. As shown in the exemplary embodiment of FIG. 1B, theanalyzer 100 includes a housing 101 which is configured to cover orretain the components of the analyzer 100 which are discussed in greaterdetail below. An opening 120 in the housing 101 is configured to receivea cassette 20. As set forth in greater detail below, the analyzer 100may also include a user interface 200 positioned within the housing 101which is configured for a user to input information into the sampleanalyzer. In this particular embodiment, the user interface 200 includesa touch screen, but as discussed below, the user interface may beconfigured differently.

FIG. 2 illustrates the sample analyzer 100 shown in FIG. 1B, except witha portion of the housing 101 and user interface 200 removed to depictsome of the other components which may be positioned within the housing101. These components will be described in greater detail below andinclude, but are not limited to a fluid flow source 40 (e.g., a vacuumsystem) configured to pressurize the cassette 20, an identificationreader 60 configured to read information associated with the cassette,and a mechanical subsystem 79 which includes a component configured tointerface with the cassette to detect the cassette within the housing.As mentioned above, an opening 120 in the housing is configured toreceive a cassette 20. As shown in FIG. 2, in one embodiment, theopening 120 is configured as an elongated slot. The opening 120 may beconfigured in this manner to receive a substantially card-shapedcassette. It should be appreciated that in other embodiments, theopening 120 may be shaped and configured differently as the invention isnot so limited.

As mentioned above, the microfluidic sample analyzer 100 may beconfigured to receive a variety of types of cassettes 20 (e.g.,microfluidic devices). FIGS. 3-9 illustrate various exemplaryembodiments of the cassette 20 for use with an analyzer 100. As shown inFIGS. 3-4 and 6, the cassette 20 may be substantially card-shaped (i.e.similar to a card key) having a substantially rigid plate-likestructure. Non-limiting examples of microfluidic systems that can bepart of cassette that can be used with the systems and methods describedherein are described in more detail in International Patent PublicationNo. WO2005/066613 (International Patent Application Serial No.PCT/US2004/043585), filed Dec. 20, 2004 and entitled “Assay Device andMethod” and U.S. patent application Ser. No. 12/113,503, published asU.S. Patent Publication No. 2008/0273918, filed May 1, 2008 and entitled“Fluidic Connectors and Microfluidic Systems”, each of which areincorporated herein by reference in their entireties for all purposes.

The cassette 20 may be configured to include a fluidic connector 220,which as shown in exemplary embodiment illustrated in FIG. 4, may snapinto one end of the cassette 20. In certain embodiments, the fluidicconnector can be used to introduce one or more fluids (e.g., a sample ora reagent) into the cassette.

In one set of embodiments, the fluidic connector is used to fluidlyconnect two (or more) channels of the cassette during first use, whichchannels are not connected prior to first use. For example, the cassettemay include two channels that are not in fluid communication prior tofirst use of the cassette. Non-connected channels may be advantageous incertain cases, such as for storing different reagents in each of thechannels. For example, a first channel may be used to store dry reagentsand a second channel may be used to store wet reagents. Having thechannels be physically separated from one another can enhance long-termstability of the reagents stored in each of the channels, e.g., bykeeping the reagent(s) stored in dry form protected from moisture thatmay be produced by reagent(s) stored in wet form. At first use, thechannels may be connected via the fluidic connector to allow fluidcommunication between the channels of the cassette. For instance, thefluidic connected may puncture seals covering inlets and/or outlets ofthe cassette to allow insertion of the fluidic connector into thecassette.

As used herein, “prior to first use of the cassette” means a time ortimes before the cassette is first used by an intended user aftercommercial sale. First use may include any step(s) requiringmanipulation of the device by a user. For example, first use may involveone or more steps such as puncturing a sealed inlet to introduce areagent into the cassette, connecting two or more channels to causefluid communication between the channels, preparation of the device(e.g., loading of reagents into the device) before analysis of a sample,loading of a sample onto the device, preparation of a sample in a regionof the device, performing a reaction with a sample, detection of asample, etc. First use, in this context, does not include manufacture orother preparatory or quality control steps taken by the manufacturer ofthe cassette. Those of ordinary skill in the art are well aware of themeaning of first use in this context, and will be able easily todetermine whether a cassette of the invention has or has not experiencedfirst use. In one set of embodiments, cassette of the invention aredisposable after first use (e.g., after completion of an assay), and itis particularly evident when such devices are first used, because it istypically impractical to use the devices at all (e.g., for performing asecond assay) after first use.

A cassette may be coupled to a fluidic connector using a variety ofmechanisms. For example, the fluidic connector may include at least onenon-fluidic feature complementary to a feature of the cassette so as toform a non-fluidic connection between the fluidic connector and thecassette upon attachment. The non-fluidic complementary feature may be,for example, a protruding feature of the fluidic connector andcorresponding complementary cavities of the cassette, which can help theuser align the fluidic connector with the cassette. In some cases, thefeature creates a substantial resistance to movement of the fluidicconnector relative to the cassette and/or alignment element upon thealignment element receiving the fluidic component (e.g., upon insertionof the fluidic component into the alignment element) and/or duringintended use of the device. The fluidic connector and/or cassette mayoptionally include one or more features such as snap features (e.g.,indentations), grooves, openings for inserting clips, zip-tiemechanisms, pressure-fittings, friction-fittings, threaded connectorssuch as screw fittings, snap fittings, adhesive fittings, magneticconnectors, or other suitable coupling mechanisms. These and otherexamples of coupling mechanisms are described in more detail in U.S.patent application Ser. No. 12/113,503, published as U.S. PatentPublication No. 2008/0273918, filed May 1, 2008 and entitled “FluidicConnectors and Microfluidic Systems”, which is incorporated herein byreference in its entirety for all purposes. Connection of the fluidicconnector to the cassette may involve forming a liquid-tight and/orair-tight seal between the components. Attachment of a fluidic connectorto a cassette may be reversible or irreversible.

As shown, the cassette 20 may be configured to include a fluidicconnector 220. In particular, the cassette 20 may include a fluidicconnector alignment element 202 which is configured to receive and matewith the connector 220. For instance, the alignment element may extendfrom the base of the cassette and comprise a cavity constructed andarranged to receive and engage the fluidic connector and therebyposition the fluidic connector in a predetermined, set configurationrelative to the base of the cassette. As shown in the illustrativeembodiments of FIG. 4, the cassette may include an alignment elementthat extends approximately perpendicular to the cassette. In otherembodiments, the alignment element may extend approximately parallel tothe cassette. Examples of alignment elements are described in moredetail in U.S. patent application Ser. No. 12/113,503, published as U.S.Patent Publication No. 2008/0273918, filed May 1, 2008 and entitled“Fluidic Connectors and Microfluidic Systems”, which is incorporatedherein by reference in its entirety for all purposes.

In some embodiments, the configuration of the alignment element and thefluidic connector may be adapted to allow insertion of the fluidicconnector into the alignment element by a sliding motion. For example,the fluidic connector may slide against one or more surfaces of thealignment element when the fluidic connector is inserted into thealignment element.

As shown in exemplary embodiment illustrated in FIG. 5, the fluidicconnector 220 may include a substantially U-shaped channel 222 which mayhold a fluid and/or reagent (e.g., a fluid sample) prior to be connectedto the cassette. Channel 222 may be housed between two shell componentswhich form the connector 220. In some embodiments, the fluidic connectormay be used to collect a sample from the patient prior to the fluidicconnector being connected to the cassette. For example, a lancet orother suitable instrument can be used to obtain a finger-stick bloodsample which may then be collected by the fluidic connector 220 andloaded into channel 222 by capillary action. In other embodiments, thefluidic connector 220 may be configured to puncture a patient's fingerto collect the sample in the channel 222. In certain embodiments, fluidconnector 220 does not contain a sample (or reagent) prior to connectionto the cassette, but simply allows fluid communication between two ormore channels of the cassette upon connection. In one embodiment, theU-shaped channel is formed with a capillary tube. The fluidic connectorcan also include other channel configurations, and in some embodiments,may include more than one channels that may be fluidically connected orunconnected to one another.

FIGS. 6-9 illustrate various exemplary embodiments of the cassette 20 ingreater detail. As shown illustratively in the exploded assembly view ofFIG. 7, the cassette 20 may include a cassette body 204 which includesat least one channel 206 configured to receive a sample or reagent andthrough which a sample or reagent may flow. The cassette body 204 mayalso include latches 208 positioned on one end that interlock with thefluidic connector alignment element 202 for a snap fit.

The cassette 20 may also include top and bottom covers 210 and 212,which may, for example, be made of a transparent material. In someembodiments, a cover can be in the form of a biocompatible adhesive andcan be made of a polymer (e.g., polyethylene (PE), a cyclic olefincopolymer (COC), polyvinyl chloride (PVC)) or an inorganic material forexample. In some cases, one or more covers are in the form of anadhesive film (e.g., a tape). For some applications, the material anddimensions of a cover are chosen such that the cover is substantiallyimpermeable to water vapor. In other embodiments, the cover can benon-adhesive, but may bond thermally to the microfluidic substrate bydirect application of heat, laser energy, or ultrasonic energy. Anyinlet(s) and/or outlet(s) of a channel of the cassette can be sealed(e.g., by placing an adhesive over the inlet(s) and/or outlet(s)) usingone or more covers. In some cases, the cover substantially seals one ormore stored reagents in the cassette.

As illustrated, the cassette body 204 may include one or more ports 214coupled to the channel 206 in the cassette body 204. These ports 214 canbe configured to align with the substantially U-shaped channel 222 inthe fluidic connector 220 when the fluidic connector 220 is coupled tothe cassette 20 to fluidly connect the channel 206 in the cassette body204 with the channel 222 in the fluidic connector 220. In certainembodiments, substantially U-shaped channel 222 can also be fluidicallyconnected to channel 207, thereby coupling channels 206 and 207. Asshown, a cover 216 may be provided over the ports 214 and the cover 216may be configured to be pieced or otherwise opened (e.g., by theconnector 220 or by other means) to fluidly connect the two channels 206and 222. Additionally, a cover 218 may be provided to cover port 219(e.g., a vacuum port) in the cassette body 204. As set forth in furtherdetail below, the port 219 may be configured to fluidly connect a fluidflow source 40 with the channel 206 to move a sample through thecassette. The cover 218 over the port 219 may be configured to bepierced or otherwise opened to fluidly connect the channel 206 with thefluid flow source 40.

The cassette body 204 may optionally include a liquid containment regionsuch as a waste area, including an absorbent material 217 (e.g., a wastepad). In some embodiments, the liquid containment region includesregions that capture one or more liquids flowing in the cassette, whileallowing gases or other fluids in the cassette to pass through theregion. This may be achieved, in some embodiments, by positioning one ormore absorbent materials in the liquid containment region for absorbingthe liquids. This configuration may be useful for removing air bubblesfrom a stream of fluid and/or for separating hydrophobic liquids fromhydrophilic liquids. In certain embodiments, the liquid containmentregion prevents liquids from passing through the region. In some suchcases, the liquid containment region may act as a waste area bycapturing substantially all of the liquid in the cassette, therebypreventing liquid from exiting the cassette (e.g., while allowing gasesto escape from an outlet of the cassette). For example, the waste areamay be used to store the sample and/or reagents in the cassette afterthey have passed through the channel 206 during the analysis of thesample. These and other arrangements may be useful when the cassette isused as a diagnostic tool, as the liquid containment region may preventa user from being exposed to potentially-harmful fluids in the cassette.Non-limiting examples of liquid containment regions are described inmore detail in U.S. patent application Ser. No. 12/196,392, published asU.S. Patent Publication No. 2009/0075390, filed Aug. 22, 2008, entitled“Liquid containment for integrated assays”, which is incorporated hereinby reference in its entirety for all purposes.

The schematic view of the cassette 20 illustrated in FIG. 8 shows oneembodiment where the cassette 20 includes a first channel 206 and asecond channel 207 spaced apart from the first channel 206. In oneembodiment, the channels 206, 207 range in largest cross-sectiondimension from approximately 50 micrometers to approximately 500micrometers, although other channel sizes and configurations may beused, as described in more detail below.

The first channel 206 may include one or more measurement zones 209 usedto analyze the sample. For example, in one illustrative embodiment, thechannel 206 includes four measurement zones 209 (e.g., connected inseries or in parallel) which are utilized during sample analysis.

In certain embodiments, one or more measurement zones are in the form ofmeandering regions (e.g., involving meandering channels), as describedin more detail below and in International Patent Publication No.WO2006/113727 (International Patent Application Serial No.PCT/US06/14583), filed Apr. 19, 2006 and entitled “Fluidic StructuresIncluding Meandering and Wide Channels”; U.S. patent application Ser.No. 12/113,503, published as U.S. Patent Publication No. 2008/0273918,filed May 1, 2008 and entitled “Fluidic Connectors and MicrofluidicSystems” and U.S. patent application Ser. No. 12/196,392, published asU.S. Patent Publication No. 2009/0075390, filed Aug. 22, 2008, entitled“Liquid containment for integrated assays”, each of which isincorporated herein by reference in their entireties for all purposes. Ameandering region may, for example, be defined by an area of at least0.25 mm², at least 0.5 mm², at least 0.75 mm², or at least 1.0 mm²,wherein at least 25%, 50%, or 75% of the area of the meandering regioncomprises an optical detection pathway. A detector that allowsmeasurement of a single signal through more than one adjacent segmentsof the meandering region may be positioned adjacent the meanderingregion. In some cases, channel 206 is fluidically connected to at leasttwo meandering regions connected in series.

As described herein, the first channel 206 and/or the second channel 207may be used to store one or more reagents used to process and analyzethe sample prior to first use of the cassette. In some embodiments, dryreagents are stored in one channel or section of a cassette and wetreagents are stored in a second channel or section of cassette.Alternatively, two separate sections or channels of a cassette may bothcontain dry reagents and/or wet reagents. Reagents can be stored and/ordisposed, for example, as a liquid, a gas, a gel, a plurality ofparticles, or a film. The reagents may be positioned in any suitableportion of a cassette, including, but not limited to, in a channel,reservoir, on a surface, and in or on a membrane, which may optionallybe part of a reagent storage area. A reagent may be associated with acassette (or components of a cassette) in any suitable manner. Forexample, reagents may be crosslinked (e.g., covalently or ionically),absorbed, or adsorbed (physisorbed) onto a surface within the cassette.In one particular embodiment, all or a portion of a channel (such as afluid path of a fluid connector or a channel of the cassette) is coatedwith an anti-coagulant (e.g., heparin). In some cases, a liquid iscontained within a channel or reservoir of a cassette prior to first useand/or prior to introduction of a sample into the cassette.

In some embodiments, the stored reagents may include fluid plugspositioned in linear order so that during use, as fluids flow to areaction site, they are delivered in a predetermined sequence. Acassette designed to perform an assay, for example, may include, inseries, a rinse fluid, a labeled-antibody fluid, a rinse fluid, and aamplification fluid, all stored therein. While the fluids are stored,they may be kept separated by substantially immiscible separation fluids(e.g., a gas such as air) so that fluid reagents that would normallyreact with each other when in contact may be stored in a common channel.

Reagents can be stored in a cassette for various amounts of time. Forexample, a reagent may be stored for longer than 1 hour, longer than 6hours, longer than 12 hours, longer than 1 day, longer than 1 week,longer than 1 month, longer than 3 months, longer than 6 months, longerthan 1 year, or longer than 2 years. Optionally, the cassette may betreated in a suitable manner in order to prolong storage. For instance,cassettes having stored reagents contained therein may be vacuum sealed,stored in a dark environment, and/or stored at low temperatures (e.g.,below 0 degrees C.). The length of storage depends on one or morefactors such as the particular reagents used, the form of the storedreagents (e.g., wet or dry), the dimensions and materials used to formthe substrate and cover layer(s), the method of adhering the substrateand cover layer(s), and how the cassette is treated or stored as awhole. Storing of a reagent (e.g., a liquid or dry reagent) in a channelmay involve sealing the inlet(s) and outlet(s) of the channel prior tofirst use or during packaging of the device.

As illustrated in the exemplary embodiment shown in FIGS. 8 and 9A-9F,channels 206 and 207 may not be in fluid communication with each otheruntil the fluidic connector 220 is coupled to the cassette 20. In otherwords, the two channels, in some embodiments, are not in fluidcommunication with one another prior to first use and/or prior tointroduction of a sample into the cassette. In particular, asillustrated, the substantially U-shaped channel 222 of the connector 220may fluidly connect the first and second channels 206, 207 such that thereagents in the second channel 207 can pass through the U-shaped channel22 and selectively move into the measurement zones 209 in the firstchannel 206. In other embodiments, the two channels 206 and 207 are influid communication with one another prior to first use, and/or prior tointroduction of a sample into the cassette, but the fluidic connectorfurther connects the two channels (e.g., to form a closed-loop system)upon first use.

In some embodiments, a cassette described herein may include one moremicrofluidic channels, although such cassettes are not limited tomicrofluidic systems and may relate to other types of fluidic systems.“Microfluidic,” as used herein, refers to a cassette, device, apparatusor system including at least one fluid channel having a maximumcross-sectional dimension of less than 1 mm, and a ratio of length tolargest cross-sectional dimension of at least 3:1. A “microfluidicchannel,” as used herein, is a channel meeting these criteria.

The “cross-sectional dimension” (e.g., a diameter) of the channel ismeasured perpendicular to the direction of fluid flow. Most fluidchannels in components of cassettes described herein have maximumcross-sectional dimensions less than 2 mm, and in some cases, less than1 mm. In one set of embodiments, all fluid channels of a cassette aremicrofluidic or have a largest cross sectional dimension of no more than2 mm or 1 mm. In another set of embodiments, the maximum cross-sectionaldimension of the channel(s) are less than 500 microns, less than 200microns, less than 100 microns, less than 50 microns, or less than 25microns. In some cases the dimensions of the channel may be chosen suchthat fluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any suitable method known to those of ordinary skill in theart. In some cases, more than one channel or capillary may be used.

A channel may include a feature on or in an article (e.g., a cassette)that at least partially directs the flow of a fluid. The channel canhave any suitable cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore.

Cassettes described herein may include channels or channel segmentspositioned on one or two sides of the cassette. In some cases, thechannels are formed in a surface of the cassette. The channel segmentsmay be connected by an intervening channel passing through the cassette.Non-limiting examples of such and other channel configurations aredescribed in more detail in U.S. patent application Ser. No. 12/640,420,filed Dec. 17, 2009, entitled, “Reagent Storage in Microfluidic Systemsand Related Articles and Methods”, which is incorporated herein byreference in its entirety for all purposes. In some embodiments, thechannel segments are used to store reagents in the device prior to firstuse by an end user. The specific geometry of the channel segments andthe positions of the channel segments within the cassettes may allowfluid reagents to be stored for extended periods of time without mixing,even during routine handling of the cassettes such as during shipping ofthe cassettes, and when the cassettes are subjected to physical shock orvibration.

In certain embodiments, a cassette includes optical elements that arefabricated on one side of a cassette opposite a series of fluidicchannels. An “optical element” is used to refer to a feature formed orpositioned on or in an article or cassette that is provided for and usedto change the direction (e.g., via refraction or reflection), focus,polarization, and/or other property of incident electromagneticradiation relative to the light incident upon the article or cassette inthe absence of the element. For example, an optical element may comprisea lens (e.g., concave or convex), mirror, grating, groove, or otherfeature formed or positioned in or on a cassette. A cassette itselfabsent a unique feature, however, would not constitute an opticalelement, even though one or more properties of incident light may changeupon interaction with the cassette. The optical elements may guideincident light passing through the cassette such that most of the lightis dispersed away from specific areas of the cassette, such asintervening portions between the fluidic channels. By decreasing theamount of light incident upon these intervening portions, the amount ofnoise in a detection signal can be decreased when using certain opticaldetection systems. In some embodiments, the optical elements comprisetriangular grooves formed on or in a surface of the cassette. The draftangle of the triangular grooves may be chosen such that incident lightnormal to the surface of the cassette is redirected at an angledependent upon the indices of refraction of the external medium (e.g.,air) and the cassette material. In some embodiments, one or more opticalelements are positioned between adjacent segments of a meandering regionof a measurement zone. Non-limiting examples of optical elements andconfigurations of channels and components with respect to the opticalelements are described in more detail in U.S. patent application Ser.No. 12/698,451, filed Feb. 2, 2010, entitled, “Structures forControlling Light Interaction with Microfluidic Devices”, which isincorporated herein by reference in its entirety for all purposes.

A cassette, or portions thereof, can be fabricated of any materialsuitable for forming a channel or other component. Non-limiting examplesof materials include polymers (e.g., polyethylene, polystyrene,polymethylmethacrylate, polycarbonate, poly(dimethylsiloxane), PVC,PTFE, PET, and a cyclo-olefin copolymer), glass, quartz, and silicon.The material forming the cassette and any associated components (e.g., acover) may be hard or flexible. Those of ordinary skill in the art canreadily select suitable material(s) based upon e.g., its rigidity, itsinertness to (e.g., freedom from degradation by) a fluid to be passedthrough it, its robustness at a temperature at which a particular deviceis to be used, its transparency/opacity to light (e.g., in theultraviolet and visible regions), and/or the method used to fabricatefeatures in the material. For instance, for injection molded or otherextruded articles, the material used may include a thermoplastic (e.g.,polypropylene, polycarbonate, acrylonitrile-butadiene-styrene, nylon 6),an elastomer (e.g., polyisoprene, isobutene-isoprene, nitrile, neoprene,ethylene-propylene, hypalon, silicone), a thermoset (e.g., epoxy,unsaturated polyesters, phenolics), or combinations thereof. Asdescribed in more detail below, cassettes including two or morecomponents or layers may be formed in different materials to tailor thecomponents to the major function(s) of the each of the components, e.g.,based upon those factors described above and herein.

In some embodiments, the material and dimensions (e.g., thickness) of acassette and/or cover are chosen such that it is substantiallyimpermeable to water vapor. For instance, a cassette designed to storeone or more fluids therein prior to first use may include a covercomprising a material known to provide a high vapor barrier, such asmetal foil, certain polymers, certain ceramics and combinations thereof.Examples of materials having low water vapor permeability are providedbelow. In other cases, the material is chosen based at least in part onthe shape and/or configuration of the cassette. For instance, certainmaterials can be used to form planar devices whereas other materials aremore suitable for forming devices that are curved or irregularly shaped.

In some instances, a cassette is comprised of a combination of two ormore materials, such as the ones listed above. For instance, channels ofthe cassette may be formed in polystyrene or other polymers (e.g., byinjection molding) and a biocompatible tape may be used to seal thechannels. The biocompatible tape or flexible material may include amaterial known to improve vapor barrier properties (e.g., metal foil,polymers or other materials known to have high vapor barriers), and mayoptionally allow access to inlets and outlets by puncturing or unpeelingthe tape. A variety of methods can be used to seal a microfluidicchannel or portions of a channel, or to join multiple layers of adevice, including but not limited to, the use of adhesives, use adhesivetapes, gluing, bonding, lamination of materials, or by mechanicalmethods (e.g., clamping, snapping mechanisms, etc.).

In some instances, a cassette comprises a combination of two or moreseparate components (e.g., layers or cassettes) mounted together.Independent channel networks (such as sections 71 and 77 of FIG. 1A),which may optionally include reagents stored therein prior to first use,may be included on or in the different components of the cassette. Theseparate components may be mounted together or otherwise associated withone another by any suitable means, such as by the methods describedherein, e.g., to form a single (composite) cassette. In someembodiments, two or more channel networks are positioned in differentcomponents or layers of the cassette and are not connected fluidicallyprior to first use, but are connected fluidically at first use, e.g., byuse of a fluidic connector. In other embodiments, the two or morechannel networks are connected fluidically prior to first use.

Advantageously, each of the different components or layers that form acomposite cassette may be tailored individually depending on thedesigned function(s) of that component or layer. For example, in one setof embodiments, one component of a composite cassette may be tailoredfor storing wet reagents. In some such embodiments, that component maybe formed in a material having a relatively low vapor permeability.Additionally or alternatively, e.g., depending on the amount of fluidsto be stored, the storage region(s) of that cassette may be made withlarger cross-sectional dimensions than channels or regions of othercomponents not used for storage of liquids. The material used to formthe cassette may be compatible with fabrication techniques suitable forforming larger cross-sectional dimensions. By contrast, a secondcomponent that may be tailored for detection of an analyte may, in someembodiments, include channel portions having smaller cross-sectionaldimensions. Smaller cross-sectional dimensions may be useful, forexample, in certain embodiments to allow more contact time betweenfluids flowing in the channel (e.g., a reagent solution or a wash fluid)and an analyte bound to a surface of the channel, for a given volume offluid. Additionally or alternatively, a channel portion of the secondcomponent may have a lower surface roughness (e.g., to increase thesignal to noise ratio during detection) compared to a channel portion ofanother component. The smaller-cross sectional dimensions or lowersurface roughness of the channel portions of the second component may,in certain embodiments, require a certain fabrication technique orfabrication tool different from that used to form a different componentof the cassette. Furthermore, in some particular embodiments, thematerial used for the second component may be well characterized forprotein attachment and detection. As such, it may be advantageous toform different channels portions used for different purposes ondifferent components of a cassette, which can then be joined togetherprior to use by an intended user. Other advantages, features ofcomponents, and examples are provided below.

FIGS. 9B-9E show a device that may include multiple components 20B and20C that are combined to form a single cassette. As shown in theseillustrative embodiments, component 20B may include a first side 21A anda second side 21B. Component 20C may include a first side 22A and asecond side 22B. Device components or parts described herein such aschannels or other entities may be formed at, on, or in the first side ofa component, a second side of a component and/or through the componentin some embodiments. For example, as shown illustratively in FIG. 9C,component 20C may include a channel 206 having an inlet and an outlet,and may be formed in a first material. Channel 206 may have any suitableconfiguration as described herein and may include, for example, one ormore reagent storage regions, measurement zones, liquid containmentregions, mixing regions, and the like. In some embodiments, channel 206is not formed through the entire thickness of component 20B. That is,the channel may be formed at or in one side of the component. Channel206 may be optionally enclosed by a cover as described herein such as atape (not shown), another component or layer of the cassette, or othersuitable component. In other embodiments, channel 206 is formed throughthe entire thickness of component 20B and covers are required on bothsides of the cassette to enclose the channel.

Component 20B may include channel 207 having an inlet and an outlet, andmay be formed in a second material, which may be the same or differentas the first material. Channel 207 may also have any suitableconfiguration as described herein, and may or may not be formed throughthe entire thickness of component 20C. Channel 207 may be enclosed byone or more covers. In some cases, the cover is not a component thatincludes one or more fluidic channels such as component 20C. Forexample, the cover may be a biocompatible tape or other surfacepositioned between components 20B and 20C. In other embodiments, channel207 may be substantially enclosed by component 20C. That is, surface 22Aof component 20C may form a portion of channel 207 as components 20B and20C lay directly adjacent to one another.

As shown illustratively in FIGS. 9D and 9E, components 20B and 20C maybe substantially planar and may lay on top of one another. In general,however, the two or more components forming a cassette can lay in anysuitable configuration with respect to one another. In some cases, thecomponents lay adjacent to one another (e.g., side by side, on top ofone another). The first components may completely overlap or onlyportions of the components may overlap with one another. For example, asshown illustratively in FIGS. 9D and 9E, component 20C may extendfurther than component 20B such that a portion of component 20C is notoverlapping or covered by component 20B. In some cases, thisconfiguration can be advantageous where component 20C is substantiallytransparent and requires light to travel through a portion of thecomponent (e.g., a reaction area, measurement zone, or detectionregion), and where component 20B is opaque or less transparent thancomponent 20C.

Furthermore, the first and second components may include any suitableshape and/or configuration. For instance, in some embodiments, the firstcomponent includes a feature complementary to a feature of the secondcomponent, so as to form a non-fluidic connection between the first andsecond components. The complementary features may, for example, aidalignment of the first and second components during assembly. Examplesof complementary features are described herein. In some cases, couplingor mating mechanisms, such as those described herein, can be used tocouple the first and second components.

The first and second components may be integrally connected to oneanother in some embodiments. As used herein, the term “integrallyconnected,” when referring to two or more objects, means objects that donot become separated from each other during the course of normal use,e.g., cannot be separated manually; separation requires at least the useof tools, and/or by causing damage to at least one of the components,for example, by breaking, peeling, or separating components fastenedtogether via adhesives or tools. Integrally connected components may beirreversibly attached to one another during the course of normal use.For example, components 20B and 20C may be integrally connected by useof an adhesive or by other bonding methods. In other embodiments, two ormore components of a cassette may be reversibly attached to one another.

As described herein, in some embodiments at least a first component anda second component forming a composite cassette may be formed indifferent materials. The system may be designed such that the firstcomponent includes a first material that aids or enhances one or morefunctionalities of the first component. For example, if the firstcomponent is designed to store a liquid reagent (e.g., in a channel ofthe component) prior to first use by a user (e.g., for at least a day, aweek, a month, or a year), the first material may be chosen to have arelatively low vapor permeability so as to reduce the amount ofevaporation of the stored liquid over time. It should be understood,however, that the same materials may be used for multiple components(e.g., layers) of a cassette in some embodiments. For instance, bothfirst and second components of a cassette may be formed in a materialhaving a low water vapor permeability.

A material used to form all or portions of a section or component of adevice may have, for example, a water vapor permeability of less thanabout 5.0 g·mm/m²·d, less than about 4.0 g·mm/m²·d, less than about 3.0g·mm/m²·d, less than about 2.0 g·mm/m²·d, less than about 1.0 g·mm/m²·d,less than about 0.5 g·mm/m²·d, less than about 0.3 g·mm/m²·d, less thanabout 0.1 g·mm/m²·d, or less than about 0.05 g·mm/m²·d. In some cases,the water vapor permeability may be, for example, between about 0.01g·mm/m²·d and about 2.0 g·mm/m²·d, between about 0.01 g·mm/m²·d andabout 1.0 g·mm/m²·d, between about 0.01 g·mm/m²·d and about 0.4g·mm/m²·d, between about 0.01 g·mm/m²·d and about 0.04 g·mm/m²·d, orbetween about 0.01 g·mm/m²·d and about 0.1 g·mm/m²·d. The water vaporpermeability may be measured at, for example, 40° C. at 90% relativehumidity (RH).

In some embodiments, a second component is not used to store a liquidprior to use by a user and may be formed in a second material having ahigher water vapor permeability than that of the first component. Forexample, the second material may have a water vapor permeability ofgreater than about 0.05 g·mm/m²·d, greater than about 0.1 g·mm/m²·d,greater than about 0.3 g·mm/m²·d, greater than about 0.5 g·mm/m²·d,greater than about 1.0 g·mm/m²·d, greater than about 2.0 g·mm/m²·d,greater than about 3.0 g·mm/m²·d, greater than about 4.0 g·mm/m²·d, orgreater than about 5.0 g·mm/m²·d.

In some cases, a first material used to form a first component of acassette has a water vapor permeability at least 1.5×, at least 2×, atleast 3×, at least 5×, at least 10×, at least 20×, at least 50×, or atleast 100× lower than that of a second material used to form a secondcomponent of a cassette.

Water vapor permeabilities of materials are known or can be determinedby those of ordinary skill in the art. Materials such as certaincyclo-olefin copolymers, for example, typically have a water vaporpermeability of less than about 0.1 g·mm/m²·d (e.g., between 0.02-0.04g·mm/m²·d), whereas certain polypropylenes have a water vaporpermeability of about 0.5 g·mm/m²·d or greater. Certain PETs have awater vapor permeability of about 1.0 g·mm/m²·d, certain PVCs have awater vapor permeability of about 1.2 g·mm/m²·d, and certainpolycarbonates have a water vapor permeability of about 4.0 g·mm/m²·d.

In some embodiments, one or more components or layers of a device may beformed in a material that makes it more suitable for processing undercertain conditions. For example, a material may be chosen in part basedon its melting temperature to allow it to be compatible with certainfabrication tools and/or methods (e.g., for forming channels of certaindimensions) such as those described herein. In some embodiments, a firstcomponent is formed in a material having a melting temperature ofgreater than about 80° C., greater than about 100° C., greater thanabout 130° C., greater than about 160° C., or greater than about 200° C.In certain embodiments, a second component designed to be combined withthe first component may be formed in a material having a meltingtemperature of less than or equal to about 200° C., less than or equalto about 160° C., less than or equal to about 130° C., less than orequal to about 100° C., or less than or equal to about 80° C. Othermelting temperatures are also possible.

In one particular set of embodiments, component 20B is formed of amaterial having a higher melting temperature than the material used toform component 20C. In one particular embodiment, a component used forstorage of a liquid reagent is formed in a material having a highermelting temperature than a material used to form another component ofthe cassette.

In certain embodiments, a cassette including first and second componentshave channel portions of different cross-sectional dimensions in each ofthe different components. As described herein, the particularcross-sectional dimensions may be chosen based in part on thefunction(s) of the channel portions, where the channel portions arepositioned relative to other parts or components of the device, andother factors.

A channel portion of a cassette may have any suitable cross-sectionaldimension. For example, a first component may include a first channelincluding at least one portion having a cross-sectional dimension of,for example, greater than about 50 microns, greater than about 100microns, greater than about 200 microns, greater than about 350 microns,greater than about 500 microns, greater than about 750 microns orgreater than about 1 mm. In some cases, a channel portion having arelatively large cross-sectional dimension may be used to store a liquidcontained therein prior to first use by a user.

In some cases, a second component of a cassette may include a secondchannel including at least one portion having a cross-sectionaldimension that is at least 1.5 times, at least 2 times, at least 3times, at least 5 times, at least 7 times or at least 10 times differentthan the cross-sectional dimension of a first channel portion of a firstcomponent of the cassette. Such differences in cross-sectionaldimensions may be due to the different functionality of the secondchannel portion in the second component compared to that of the firstcomponent. The second channel of the second component may include atleast one portion having a cross-sectional dimension of, for example,less than about 1 mm, less than about 750 microns, less than about 500microns, less than about 350 microns, less than about 200 microns, lessthan about 100 microns or less than about 50 microns. For example, insome cases, a channel having a relatively smaller cross-sectionaldimension than that of a first channel of a first component may besuitable for a detection region of the device, for controlling rates offluid flow, or for other purposes.

In some embodiments, channel portions in different components of acassette have different cross-sectional dimensions and are formed inmaterials having different melting temperatures. For example, in someinstances, a channel portion having a relatively small cross-sectionaldimension (e.g., less than about 300 microns, less than about 200microns, or less than about 100 microns) may be formed in a materialhaving a relatively low melting temperature (e.g., less than about 100°C.), whereas a channel portion having a relatively largercross-sectional dimension (e.g., greater than about 100 microns, greaterthan about 200 microns, or greater than about 300 microns) may be formedin a material having a relatively higher melting temperature (e.g.,greater than about 100° C.).

In certain cases, channels from different components or layers of adevice may have different surface roughness. For example, a channel thatis designed to be part of a detection region may have a lower surfaceroughness than a channel that is not used in a detection process or isused in a detection process that requires less sensitivity. Substantialroughness on the surface of a channel portion may result in unwantedscattering or redirection of light at an undesired angle. Channels fromdifferent components or layers of a device having different surfaceroughness may be advantageous because a channel having a relatively lowsurface roughness may be more complicated and/or more expensive tofabricate than a channel having a higher surface roughness. For example,certain fabrication tools for molding made by micromachining orlithography techniques have less surface roughness (and, therefore, formchannel portions having less surface roughness) compared to tools madeby machining, but may be more complicated and/or expensive to fabricate.

In some embodiments, at least a portion of a first channel of a firstcomponent may have a root mean square surface (RMS) roughness of lessthan about less than or equal to about 10 microns. In certainembodiments, the RMS surface roughness may be, for example, less than orequal to about 5 microns, less than or equal to about 3 microns, lessthan or equal to about 1 micron, less than or equal to about 0.8microns, less than or equal to about 0.5 microns, less than or equal toabout 0.3 microns, or less than or equal to about 0.1 microns. RMSsurface roughness is a term known to those skilled in the art, and maybe expressed as:

$\sigma_{h} = {\left\lbrack {\langle\left( {z - z_{m}} \right)^{2}\rangle} \right\rbrack^{1/2} = \left\lbrack {\frac{1}{A}{\int_{A}{\left( {z - z_{m}} \right)^{2}\ {A}}}} \right\rbrack^{1/2}}$

where A is the surface to be examined, and |z−z_(m)| is the local heightdeviation from the mean.

At least a portion of a second channel of a second component may have,for example, a root mean square surface roughness different from that ofthe first component. The second channel portion may have a RMS surfaceroughness of, for example, greater than about 0.1 microns, greater thanabout 0.3 microns, greater than about 0.5 micron, greater than about 1micron, greater than about 3 microns, greater than about 5 microns, orgreater than about 10 microns.

In certain embodiments, first and second components of a cassette havedifferent degrees of optical clarity. For example, a first component maybe substantially opaque, and a second component may be substantiallytransparent. The substantially transparent component may be suitable foroptical detection of a sample or analyte contained within the component.

In one set of embodiments, a material used form a component (e.g., afirst or a second component) of a cassette has an optical transmissionof greater than 90% between 400 and 800 nm wavelengths of light (e.g.,light in the visible range). Optical transmission may be measuredthrough a material having a thickness of, for example, about 2 mm (or inother embodiments, about 1 mm or about 0.1 mm) In some instances, theoptical transmission is greater than 80%, greater than 85%, greater than88%, greater than 92%, greater than 94%, or greater than 96% between 400and 800 nm wavelengths of light. Another component of the device may beformed in a material having an optical transmission of less than 96%,less than 94%, less than 92%, less than 90%, less than 85%, less than80%, less than 50%, less than 30%, or less than 10% between 400 and 800nm wavelengths of light.

As described herein, different components or layers of a device mayinclude channels made by different (or the same) fabrication toolsand/or methods. For instance, injection molding may be used to form onecomponent, and a different technique (e.g., machining) may be used toform another component. In another example, a first channel portion of afirst component may be formed by a molding (e.g., injection molding)process involving the use of a fabrication tool made by milling or by alithography process. In some cases, channel portions formed by afabrication tool made by milling may have a substantially roundedcross-sectional area, whereas channel portions formed by fabricationtool made by a lithography process may have a substantially trapezoidalcross-sectional area. Other methods for forming channel portions havingsubstantially rounded cross-sectional areas, substantially trapezoidalcross-sectional areas, or cross-sectional shapes, are also possible.Advantages and further description of channel portions having differentcross-sectional shapes are described in more detail in U.S. patentapplication Ser. No. 12/640,420, published as U.S. Patent PublicationNo. 2010/0158756, filed Dec. 17, 2009, entitled, “Reagent Storage inMicrofluidic Systems and Related Articles and Methods”, which isincorporated herein by reference in its entirety. A second channelportion of a second component may be formed using a fabrication toolmade by the same or a different method, and/or may have the same ordifferent cross-sectional shape compared to a channel portion of a firstcomponent.

As described herein, in some embodiments a channel of a first componentof a cassette is not in fluid communication with a channel of a secondcomponent of a cassette prior to first use by a user. For instance, evenafter mating of the two components, as shown illustratively in FIG. 9D,channels 206 and 207 are not in fluid communication with one another.However, the cassette may further include other parts or components suchas fluidic connector alignment element 202 (FIG. 9E), which can attachto first and/or second components 20B and 20C or to other portions ofthe cassette. As described herein, the fluidic connector alignmentelement may be configured to receive and mate with fluidic connector220, which can allow fluid communication between channels 206 and 207 ofthe first and second components, respectively. For example, the fluidicconnector may include a fluid path including a fluid path inlet and afluid path outlet, wherein the fluid path inlet can be fluidicallyconnected to the outlet of channel 206 and the fluid path outlet can befluidically connected to the inlet of channel 207 (or vice versa). Thefluid path of the fluidic connector may have any suitable length (e.g.,at least 1 cm, at least 2 cm, at least 3 cm, at least 5 cm) forconnecting the channels. The fluidic connector may be a part of a kitalong with a cassette, and packaged such that the fluidic connector isnot fluidically connecting channels 206 and 207.

A fluidic connector may have any suitable configuration with respect toa cassette, or components of a cassette. As shown illustratively in FIG.9E, upon connection of the fluidic connector to the cassette, thefluidic connector may be positioned on a side of a component (e.g.,component 20B) opposite another component (e.g., component 20C). Inother embodiments, a fluidic connector can be positioned between twocomponents of a cassette. For instance, the fluidic connector may be acomponent or layer positioned between (e.g., sandwiched between) twocomponents of the cassette. Other configurations are also possible.

Additionally, a fluidic connector may lie substantially perpendicular toone or more components or layers of a cassette, e.g., as shownillustratively in FIG. 9E. In other embodiments, a fluidic connector maylie substantially parallel to (e.g., on top of or flat against) one ormore components of a cassette. Other configurations are also possible.

In some cases, an alignment element and/or a fluidic connector isphysically connected to only a single component of a multi-componentcassette, while in other cases, an alignment element and/or a fluidicconnector is physically connected to multiple components of amulti-component cassette. In certain embodiments, a portion of acomponent of the cassette that is physically connected to an alignmentelement and/or a fluidic connector has a certain thickness to allowsuitable connection. For example, where the fluidic connector isdesigned to be inserted into an inlet and an outlet of channels of acassette, the cassette at the insertion region may have a certain (e.g.,minimal) thickness. The cassette, or one or more components of acassette, at a region designed for connection with a fluidic connectormay be, for example, have a thickness of at least 1 cm, at least 1.5 cm,at least 2 cm, at least 2.5, at least 3 cm, at least 4 cm, or at least 5cm. Other portions of the cassette (or components of the cassette) notdesigned for connection with an alignment element and/or a fluidicconnector may have a thickness of, for example, less than 5 cm, lessthan 4 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than1.5 cm, less than 1 cm, less than 0.5 cm, or less than 0.1 cm.

Although much of the description herein is directed towards a cassettehaving one or more components or layers including channel networks, inother embodiments, a cassette may include more than 2, more than 3, ormore than 4 such components or layers. For example, as shownillustratively in FIG. 9F, a cassette may include components 20B, 20C,20D, and 20E, each including at least one channel or network ofchannels. In some instances, the channel(s) of one or more components(e.g., 2, 3, or all components) may be fluidically unconnected prior tofirst use, but may be connected fluidically at first use, e.g., by useof a fluidic connector. In other embodiments, the channel(s) of one ormore components (e.g., 2, 3, or all components) are connectedfluidically prior to first use.

As described herein, each of the components or layers of a cassette maybe designed to have a specific function that is different from afunction of another component of the cassette. In other embodiments, twoor more components may have the same function. For example, as shown inthe illustrative embodiment of FIG. 9F, each of components 20C, 20D and20E may have multiple measurement zones 209 connected in series. Uponconnection of fluidic connector 222 to the composite cassette, portionsof a sample (or multiple samples) may be introduced into the channelnetwork in each of components 20C, 20D and 20E to perform multipleanalyses.

In some embodiments, at least first and second components of a cassettemay be a part of a device or a kit used for determining a particularchemical or biological condition. The device or kit may include, forexample, a first component comprising a first channel in a firstmaterial, the first channel including an inlet, an outlet and, betweenthe first inlet and outlet, at least one portion having across-sectional dimension greater than 200 microns. The device or kitmay also include a second component comprising a second channel in asecond material, the second channel including an inlet, an outlet and,between the second inlet and outlet, at least one portion having across-sectional dimension less than 200 microns. In some cases, thedevice or kit is packaged such that the first and second components areconnected to one another. For example, the first and second componentsmay be integrally connected to one another. In other embodiments, thefirst and second components are reversibly attached to one another. Thedevice or kit may further include a fluidic connector for fluidicallyconnecting the first and second channels, the fluidic connectorcomprising a fluid path, including a fluid path inlet and a fluid pathoutlet, wherein the fluid path inlet can be fluidically connected to theoutlet of the first channel and the fluid path outlet can be fluidicallyconnected to the inlet of the second channel. In some embodiments, thedevice or kit is packaged such that the fluidic connector is notfluidically connecting the first and second channels in the package.Upon first use of the device by an intended user, the fluidic connectorcan be used to bring the first and second channels into fluidcommunication with one another.

As described herein, a device or kit may include channel portions ondifferent components of a cassette that may differ from one another. Assuch, in certain embodiments, a device comprises one or more of thefollowing features: the first material used to form a first channelportion of a first component is different from a second material used toform a second channel portion of a second component; the first channelportion has a different cross-sectional shape from that of the secondchannel portion; and/or the first channel portion has a different RMSsurface roughness than that of the second channel portion. Channelportions may also have other differences as described herein.

A cassette described herein may have any suitable volume for carryingout an analysis such as a chemical and/or biological reaction or otherprocess. The entire volume of a cassette includes, for example, anyreagent storage areas, measurement zones, liquid containment regions,waste areas, as well as any fluid connectors, and fluidic channelsassociated therewith. In some embodiments, small amounts of reagents andsamples are used and the entire volume of the fluidic device is, forexample, less than 10 mL, 5 mL, 1 mL, 500 μL, 250 μL, 100 μL, 50 μL, 25μL, 10 μL, 5 μL, or 1 μL.

A cassette described herein may be portable and, in some embodiments,handheld. The length and/or width of the cassette may be, for example,less than or equal to 20 cm, 15 cm, 10 cm, 8 cm, 6 cm, or 5 cm. Thethickness of the cassette may be, for example, less than or equal to 5cm, 3 cm, 2 cm, 1 cm, 8 mm, 5 mm, 3 mm, 2 mm, or 1 mm Advantageously,portable devices may be suitable for use in point-of-care settings.

It should be understood that the cassettes and their respectivecomponents described herein are exemplary and that other configurationsand/or types of cassettes and components can be used with the systemsand methods described herein.

The methods and systems described herein may involve variety ofdifferent types of analyses, and can be used to determine a variety ofdifferent samples. In some cases, an analysis involves a chemical and/orbiological reaction. In some embodiments, a chemical and/or biologicalreaction involves binding. Different types of binding may take place incassettes described herein. Binding may involve the interaction betweena corresponding pair of molecules that exhibit mutual affinity orbinding capacity, typically specific or non-specific binding orinteraction, including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc. Binding may also occur between proteins or othercomponents and cells. In addition, devices described herein may be usedfor other fluid analyses (which may or may not involve binding and/orreactions) such as detection of components, concentration, etc.

In some cases, a heterogeneous reaction (or assay) may take place in acassette; for example, a binding partner may be associated with asurface of a channel, and the complementary binding partner may bepresent in the fluid phase. Other solid-phase assays that involveaffinity reaction between proteins or other biomolecules (e.g., DNA,RNA, carbohydrates), or non-naturally occurring molecules, can also beperformed. Non-limiting examples of typical reactions that can beperformed in a cassette include chemical reactions, enzymatic reactions,immuno-based reactions (e.g., antigen-antibody), and cell-basedreactions.

Non-limiting examples of analytes that can be determined (e.g.,detected) using cassettes described herein include specific proteins,viruses, hormones, drugs, nucleic acids and polysaccharides;specifically antibodies, e.g., IgD, IgG, IgM or IgA immunoglobulins toHTLV-I, HIV, Hepatitis A, B and non A/non B, Rubella, Measles, HumanParvovirus B19, Mumps, Malaria, Chicken Pox or Leukemia; human andanimal hormones, e.g., thyroid stimulating hormone (TSH), thyroxine(T4), luteinizing hormone (LH), follicle-stimulating hormones (FSH),testosterone, progesterone, human chorionic gonadotropin, estradiol;other proteins or peptides, e.g. troponin I, c-reactive protein,myoglobin, brain natriuretic protein, prostate specific antigen (PSA),free-PSA, complexed-PSA, pro-PSA, EPCA-2, PCADM-1, ABCA5, hK2, beta-MSP(PSP94), AZGP1, Annexin A3, PSCA, PSMA, JM27, PAP; drugs, e.g.,paracetamol or theophylline; marker nucleic acids, e.g., PCA3,TMPRS-ERG; polysaccharides such as cell surface antigens for HLA tissuetyping and bacterial cell wall material. Chemicals that may be detectedinclude explosives such as TNT, nerve agents, and environmentallyhazardous compounds such as polychlorinated biphenyls (PCBs), dioxins,hydrocarbons and MTBE. Typical sample fluids include physiologicalfluids such as human or animal whole blood, blood serum, blood plasma,semen, tears, urine, sweat, saliva, cerebro-spinal fluid, vaginalsecretions; in-vitro fluids used in research or environmental fluidssuch as aqueous liquids suspected of being contaminated by the analyte.

In some embodiments, one or more reagents that can be used to determinean analyte of a sample (e.g., a binding partner of the analyte to bedetermined) is stored in a channel or chamber of a cassette prior tofirst use in order to perform a specific test or assay. In cases wherean antigen is being analyzed, a corresponding antibody or aptamer can bethe binding partner associated with a surface of a microfluidic channel.If an antibody is the analyte, then an appropriate antigen or aptamermay be the binding partner associated with the surface. When a diseasecondition is being determined, it may be preferred to put the antigen onthe surface and to test for an antibody that has been produced in thesubject. Such antibodies may include, for example, antibodies to HIV.

In some embodiments, a cassette is adapted and arranged to perform ananalysis involving accumulating an opaque material on a region of amicrofluidic channel, exposing the region to light, and determining thetransmission of light through the opaque material. An opaque materialmay include a substance that interferes with the transmittance of lightat one or more wavelengths. An opaque material does not merely refractlight, but reduces the amount of transmission through the material by,for example, absorbing or reflecting light. Different opaque materialsor different amounts of an opaque material may allow transmittance ofless than, for example, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 1 percentof the light illuminating the opaque material. Examples of opaquematerials include molecular layers of metal (e.g., elemental metal),ceramic layers, polymeric layers, and layers of an opaque substance(e.g., a dye). The opaque material may, in some cases, be a metal thatcan be electrolessly deposited. These metals may include, for example,silver, copper, nickel, cobalt, palladium, and platinum.

An opaque material that forms in a channel may include a series ofdiscontinuous independent particles that together form an opaque layer,but in one embodiment, is a continuous material that takes on agenerally planar shape. The opaque material may have a dimension (e.g.,a width of length) of, for example, greater than or equal to 1 micron,greater than or equal to 5 microns, greater than 10 microns, greaterthan or equal to 25 microns, or greater than or equal to 50 microns. Insome cases, the opaque material extends across the width of the channel(e.g., a measurement zone) containing the opaque material. The opaquelayer may have a thickness of, for example, less than or equal to 10microns, less than or equal to 5 microns, less than or equal to 1micron, less than or equal to 100 nanometers or less than or equal to 10nanometers. Even at these small thicknesses, a detectable change intransmittance can be obtained. The opaque layer may provide an increasein assay sensitivity when compared to techniques that do not form anopaque layer.

In one set of embodiments, a cassette described herein is used forperforming an immunoassay (e.g., for human IgG or PSA) and, optionally,uses silver enhancement for signal amplification. A cassette describedherein may have one or more similar characteristics as those describedin U.S. patent application Ser. No. 12/113,503, published as U.S. PatentPublication No. 2008/0273918, filed May 1, 2008 and entitled “FluidicConnectors and Microfluidic Systems”, which is incorporated herein byreference. In such an immunoassay, after delivery of a sample containinghuman IgG to a reaction site or analysis region, binding between thehuman IgG and anti-human IgG can take place. One or more reagents, whichmay be optionally stored in a channel of the device prior to use, canthen flow over this binding pair complex. One of the stored reagents mayinclude a solution of metal colloid (e.g., a gold conjugated antibody)that specifically binds to the antigen to be detected (e.g., human IgG).This metal colloid can provide a catalytic surface for the deposition ofan opaque material, such as a layer of metal (e.g., silver), on asurface of the analysis region. The layer of metal can be formed byusing a two component system: a metal precursor (e.g., a solution ofsilver salts) and a reducing agent (e.g., hydroquinone,chlorohydroquinone, pyrogallol, metol, 4-aminophenol and phenidone),which can optionally be stored in different channels prior to use.

As a positive or negative pressure differential is applied to thesystem, the silver salt and reducing solutions can merge at a channelintersection, where they mix (e.g., due to diffusion) in a channel, andthen flow over the analysis region. Therefore, if antibody-antigenbinding occurs in the analysis region, the flowing of the metalprecursor solution through the region can result in the formation of anopaque layer, such as a silver layer, due to the presence of thecatalytic metal colloid associated with the antibody-antigen complex.The opaque layer may include a substance that interferes with thetransmittance of light at one or more wavelengths. An opaque layer thatis formed in the channel can be detected optically, for example, bymeasuring a reduction in light transmittance through a portion of theanalysis region (e.g., a serpentine channel region) compared to aportion of an area that does not include the antibody or antigen.Alternatively, a signal can be obtained by measuring the variation oflight transmittance as a function of time, as the film is being formedin an analysis region. The opaque layer may provide an increase in assaysensitivity when compared to techniques that do not form an opaquelayer. Additionally, various amplification chemistries that produceoptical signals (e.g., absorbance, fluorescence, glow or flashchemiluminescence, electrochemiluminescence), electrical signals (e.g.,resistance or conductivity of metal structures created by an electrolessprocess) or magnetic signals (e.g., magnetic beads) can be used to allowdetection of a signal by a detector.

Various types of fluids can be used with the cassettes described herein.As described herein, fluids may be introduced into the cassette at firstuse, and/or stored within the cassette prior to first use. Fluidsinclude liquids such as solvents, solutions and suspensions. Fluids alsoinclude gases and mixtures of gases. When multiple fluids are containedin a cassette, the fluids may be separated by another fluid that ispreferably substantially immiscible in each of the first two fluids. Forexample, if a channel contains two different aqueous solutions, aseparation plug of a third fluid may be substantially immiscible in bothof the aqueous solutions. When aqueous solutions are to be keptseparate, substantially immiscible fluids that can be used as separatorsmay include gases such as air or nitrogen, or hydrophobic fluids thatare substantially immiscible with the aqueous fluids. Fluids may also bechosen based on the fluid's reactivity with adjacent fluids. Forexample, an inert gas such as nitrogen may be used in some embodimentsand may help preserve and/or stabilize any adjacent fluids. An exampleof an substantially immiscible liquid for separating aqueous solutionsis perfluorodecalin. The choice of a separator fluid may be made basedon other factors as well, including any effect that the separator fluidmay have on the surface tension of the adjacent fluid plugs. It may bepreferred to maximize the surface tension within any fluid plug topromote retention of the fluid plug as a single continuous unit undervarying environmental conditions such as vibration, shock andtemperature variations. Separator fluids may also be inert to anreaction site (e.g., measurement zone) to which the fluids will besupplied. For example, if a reaction site includes a biological bindingpartner, a separator fluid such as air or nitrogen may have little or noeffect on the binding partner. The use of a gas (e.g., air) as aseparator fluid may also provide room for expansion within a channel ofa fluidic device should liquids contained in the device expand orcontract due to changes such as temperature (including freezing) orpressure variations.

As set forth in greater detail below, the microfluidic sample analyzer100 may include a fluid flow source 40 (e.g., a pressure-control system)which may be fluidly connected to the channels 206, 207, 222 topressurize the channels to move the sample and/or other reagents throughthe channels. In particular, the fluid flow source 40 may be configuredto move a sample and/or reagent initially from the substantiallyU-shaped channel 222 into the first channel 206. The fluid flow source40 may also be used to move the reagents in the second channel 207through the substantially U-shaped channel 222 and into the firstchannel 206. After the sample and reagents pass through the measurementzones 209 and are analyzed, the fluid flow source 40 may be configuredto move the fluids into the absorbent material 217 of the cassette 200.In one embodiment, the fluid flow source is a vacuum system. It shouldbe understood, however, that other sources of fluid flow such as valves,pumps, and/or other components can be used.

The top view of a cassette 20 in FIG. 9A illustrates many of thecomponents discussed above, except that in this embodiment, the channels206, 207 within the cassette housing are configured differently than inthe schematic view shown in FIG. 8. In one embodiment, the cassettehousing includes at least one surface configured to interact with amicrofluidic sample analyzer such that the cassette may be inserted intoand retained within the analyzer. In one embodiment, as illustrated inFIG. 9A, the housing includes a cammed surface along a side portion ofthe cassette 20. In this particular embodiment, the cammed surfaceincludes a notch 230 formed at one end of the cassette 20. The other endof the cassette 20 includes a curved corner surface 232. As set forth ingreater detail below, this cammed surface of the cassette may beconfigured to interact with the sample analyzer 100 such that theanalyzer 100 can detect the presence of the cassette 20 within thehousing 10 and/or position the cassette 20 within the analyzer 100. Inparticular, the curved corner surface may be configured to contact aportion of the analyzer, such as an arm, and the notch may be configuredto retain that portion of the analyzer to retain the cassette within theanalyzer.

Turning now to FIGS. 10-12, internal components of the sample analyzer100 will now be discussed. In the sub-assembly views of FIGS. 10-12, acassette 20 is shown inserted into the opening 120 in the housing. Inone embodiment, the housing 101 includes a component configured tointerface with and engage a mating component on the cassette. In thisparticular illustrated embodiment, the housing 101 includes an arm 121positioned within the housing that is configured to engage the cammedsurface on the cassette discussed above. In a first position, the arm121 extends at least partially into the opening 120 in the housing suchthat as the cassette 20 is inserted into the opening 120, the arm ispushed away from the opening 120 into a second position allowing thecassette 20 to enter the opening 120.

The arm 121 may be configured to contact a side surface of the cassetteas the cassette is inserted into the opening 120. As shown in FIG. 9A,the end of the cassette 20 may include a curved surface 232 which mayprovide a smooth transition for the arm 121 to contact the side surfaceof the cassette. In one embodiment, the arm 121 is coupled to a spring122 to make the arm spring loaded such that it will press against theside surface of the cassette when the cassette is within the analyzer100. In particular, the spring loaded arm is urged back towards and, incertain embodiments essentially into the first position. In oneembodiment, the end of the arm 121 includes a roller 124 which engagesthe side surface of the cassette 20. The roller 124 may be configured tominimize friction between the two components as the cassette is insertedinto position. As shown in FIGS. 11 and 12, once the arm 121 engages thenotch 230, the arm 121 is pushed back out to its first position due tothe bias of the spring 122. Once the arm 121 engages this inwardlycammed surface, the cassette 20 is positioned and retained within thehousing 10 of the analyzer 100, and the bias of the spring 122 preventsthe cassette 20 from slipping out of the analyzer.

It should be appreciated that the spring loaded arm 121 in the analyzer100 and the notch 230 on the cassette 20 may be configured to detect andposition the cassette 20 in the analyzer. As set forth below, it shouldalso be recognized that this arrangement may help to indicate to a userthat the cassette 20 is positioned correctly in the analyzer and isready to be analyzed and processed. After the analysis is conducted, auser may remove the cassette 20 from the analyzer 100 by pulling thecassette 20 out of the opening 120. The user may either exert a forcethat would overcome the bias of the spring 122 and/or the analyzer 100may be configured with an unlocking mechanism (not shown) to move thearm 121 into its second position such that it is no longer in contactwith the notch 230 on the cassette 20.

In one embodiment, an electronic position switch indicates when theroller 124 on the arm 121 is pushed out into its second position (e.g.,as the cassette 20 is being inserted into the analyzer). The electronicposition switch may also indicate when the roller returns backtowards/to its first position (e.g., either when there is no cassettewithin the analyzer or when the cassette is fully inserted into theanalyzer and the roller is engaged within the notch 230). Anotherposition switch may be configured to indicate when the cassette is fullyinserted and accurately positioned within the analyzer 100. Thus, theswitch may be used to indicate to a user that the cassette 20 ispositioned correctly in the analyzer. These various cassette sensors 410are shown in the schematic diagram shown in FIG. 16, which is discussedin greater detail below.

Many mechanical and electro-mechanical techniques can be used toreliably load and unload the cassette. For example, a tray, such as thatfound in a CD player, may hold a cassette and slide in and out of theanalyzer. This sliding can be accomplished by hand or by a motor (e.g.,directly driving the tray or by use of pulleys). Another example ofloading and unloading the cassette includes the use of a motorized unitthat is in physical contact with the cassette. For instance, themotorized unit may mate with the cassette through friction one or moresides and/or a top and/or bottom of the cassette. In some cases, theunit may mate with the cassette with gear teeth fabricated on the sideof the cassette.

FIG. 10 also illustrates a portion of a fluid flow source 40 which maybe configured to pressurize the channel 206 (and channel 207 if fluidlyconnected to 206) in the cassette 20 to move a sample through thechannel. FIG. 15 also illustrates a fluid flow source 40. In oneillustrative embodiment, the fluid flow source 40 is a vacuum system andincludes a vacuum source or pump 42, two vacuum reservoirs 44, 45 whichmay be separated by a vacuum regulator 46 and a manifold 48 to provide afluid connection between the vacuum reservoirs 44 and the cassette 20.The manifold 48 may also include one or more fluid connections to one ormore ports on the cassette. For example, the manifold may provide afluidic connection between port 213 and a valve (such as a solenoidvalve). Opening and closing this valve may control where air can enterthe cassette, thus serving as a vent valve in certain embodiments.

As mentioned above, in one embodiment, the vacuum source 42 is a pump,such as a solenoid operated diaphragm pump. In other embodiments, fluidflow may be driven/controlled via use of other types of pumps or sourcesof fluid flow. For example, in one embodiment, a syringe pump may beused to create a vacuum by pulling the syringe plunger in an outwarddirection. In other embodiments, a positive pressure is applied to oneor more inlets of the cassette to provide a source of fluid flow.

In some embodiments, fluid flow takes place while applying asubstantially constant non-zero pressure drop (i.e., ΔP) across an inletand an outlet of a cassette. In one set of embodiments, an entireanalysis is performed while applying a substantially constant non-zeropressure drop (i.e., ΔP) across an inlet and an outlet of a cassette. Asubstantially constant non-zero pressure drop can be achieved, forexample, by applying a positive pressure at the inlet or a reducedpressure (e.g., a vacuum) at the outlet. In some cases, a substantiallyconstant non-zero pressure drop is achieved while fluid flow does nottake place predominately by capillary forces and/or without the use ofactuating valves (e.g., without changing a cross-sectional area of achannel of a fluid path of the cassette). In some embodiments, duringessentially the entire analysis conducted in the cassette, asubstantially constant non-zero pressure drop may be present across, forexample, an inlet to a measurement zone (which may be connected to afluidic connector) and an outlet downstream of the measurement zone(e.g., an outlet downstream of a liquid containment region),respectively.

In one embodiment, a vacuum source is configured to pressurize a channelto approximately −60 kPa (approximately ⅔ atmosphere). In anotherembodiment, the vacuum source is configured to pressurize a channel toapproximately −30 kPa. In certain embodiments, a vacuum sources isconfigured to pressurize a channel to, for example, between −100 kPa and−70 kPa, between −70 kPa and −50 kPa, between −50 kPa and −20 kPa, orbetween −20 kPa and −1 kPa.

As mentioned above, in one embodiment, two vacuum reservoirs 44, 45 maybe provided. The pump may be turned on such that the first reservoir 44may be pressurized to approximately −60 kPa. A regulator 46 positionedbetween reservoir 44 and 45 may ensure that the second reservoir 45 mayonly be pressurized to a different pressure, for example, approximately−30 kPa. This regulator may maintain the pressure of reservoir 45 at −30kPa (or at another suitable pressure) as long as reservoir 44 remainedat a certain pressure range, e.g., between −60 kPa and −30 kPa. Pressuresensors may monitor the pressure within each reservoir 44, 45. If thepressure in the first reservoir 44 reaches a set point (for example,approximately −40 kPa), the pump may be actuated to decrease thepressure in the first reservoir 44. The second reservoir 45 may beconfigured to detect any leaks in the overall vacuum system 40. As shownin FIG. 15, the vacuum system 40 may include a filter 58 coupled to thereservoirs 44, 45. A solenoid valve 59 is shown which serves as the ventvalve connected through the manifold to port 213.

Once the cassette 20 is positioned within the analyzer 100, the fluidflow source 40 may be coupled to the cassette 20 to ensure a fluid-tightconnection. As mentioned above, the cassette 20 may include a port 219configured to couple the channel 206, and channel 207 if fluidicallyconnected to 206, with the fluid flow source 40. As shown in FIG. 14, inone embodiment, seals, or o-rings 52 are positioned around the port 219and a linear solenoid 50 may be positioned above the o-rings 52 to pressand seal the o-rings against the cassette body 200. As shown in FIG. 14,a manifold adapter 54 may be positioned between the linear solenoid 50and the manifold 48, and passive return springs 56 may be providedaround the manifold 48 to urge the manifold away from the cassette body200 when the solenoid is not charged. In one embodiment, multiple portson the cassette 20 may interface with the manifold 48. For example, asshown in the exemplary embodiment illustrated in FIG. 9A, in addition tothe port 219, there may be two venting ports 215 and a mixing port 213.The interface between each port and the manifold may be independent(e.g., there may be no fluidic connection inside the manifold).

In one embodiment, when the fluid flow source 40 is activated, thechannel 206, 207 in the cassette 20 may be pressurized (e.g., toapproximately −30 kPa) which will drive the fluids within the channel(both fluid sample as well as reagents) toward the outlet. In anembodiment which includes the vent ports 215 and the mixing port 213, avent valve 59 connected to port 213 through the manifold 48 mayinitially be open which may enable all of the reagents downstream of themixing port 213 to move toward the outlet, but will not cause reagentsupstream of the mixing port 213 to move. Configurations and uses of ventvalves are described in more detail in U.S. Patent Apl. Ser. No.61/263,981, filed Nov. 14, 2009 and entitled, “Fluid Mixing and Deliveryin Microfluidic Systems, which is incorporated herein by reference inits entirety for all purposes. Once the vent valve is closed, reagentsupstream of the mixing port 213 may move toward a mixing port and thento the outlet. For example, fluids can be stored serially in a channelupstream of the mixing port, and after closing a vent valve positionedalong the channel, the fluids can flow sequentially towards the channeloutlet. In some cases, fluids can be stored in separate, intersectingchannels, and after closing a vent valve the fluids will flow togethertoward a point of intersection. This set of embodiments can be used, forexample, to controllably mix the fluids as they flow together. Thetiming of delivery and the volume of fluid delivered can be controlled,for example, by the timing of the vent valve actuation.

Advantageously, vent valves can be operated without constricting thecross-section of the microfluidic channel on which they operate, asmight occur with certain valves in the prior art. Such a mode ofoperation can be effective in preventing leaking across the valve.Moreover, because vent valves can be used, some systems and methodsdescribed herein do not require the use of certain internal valves,which can be problematic due to, for example, their high expense,complexity in fabrication, fragility, limited compatibility with mixedgas and liquid systems, and/or unreliability in microfluidic systems.

It should be understood that while vent valves are described, othertypes of valving mechanisms can be used with the systems and methodsdescribed herein. Non-limiting examples of a valving mechanism which maybe operatively associated with a valve include a diaphragm valve, ballvalve, gate valve, butterfly valve, globe valve, needle valve, pinchvalve, poppet valve, or pinch valve. The valving mechanism may beactuated by any suitable means, including a solenoid, a motor, by hand,by electronic actuation, or by hydraulic/pneumatic pressure.

As previously mentioned, all of the liquids in the cassette 20 (sampleand reagents) may move into the liquid containment area which mayinclude an absorbent material 217. In one embodiment, the absorbentmaterial absorbs only liquids such that gases may flow out of thecassette through the outlet.

A variety of determination (e.g., measuring, quantifying, detecting, andqualifying) techniques may be used, e.g., to analyze a sample componentor other component or condition associated with a microfluidic system orcassette described herein. Determination techniques may includeoptically-based techniques such as light transmission, light absorbance,light scattering, light reflection and visual techniques. Determinationtechniques may also include luminescence techniques such asphotoluminescence (e.g., fluorescence), chemiluminescence,bioluminescence, and/or electrochemiluminescence. In other embodiments,determination techniques may measure conductivity or resistance. Assuch, an analyzer may be configured to include such and other suitabledetection systems.

Different optical detection techniques provide a number of options fordetermining reaction (e.g., assay) results. In some embodiments, themeasurement of transmission or absorbance means that light can bedetected at the same wavelength at which it is emitted from a lightsource. Although the light source can be a narrow band source emittingat a single wavelength it may also may be a broad spectrum source,emitting over a range of wavelengths, as many opaque materials caneffectively block a wide range of wavelengths. In some embodiments, asystem may be operated with a minimum of optical devices (e.g., asimplified optical detector). For instance, the determining device maybe free of a photomultiplier, may be free of a wavelength selector suchas a grating, prism or filter, may be free of a device to direct orcolumnate light such as a columnator, or may be free of magnifyingoptics (e.g., lenses). Elimination or reduction of these features canresult in a less expensive, more robust device.

FIGS. 10-14 illustrate an exemplary optical system 80 which may bepositioned in the housing 10 of the analyzer 100. As shownillustratively in these embodiments, the optical system 80 includes atleast a first light source 82 and a detector 84 spaced apart from thefirst light source. The first light source 82 may be configured to passlight through a first measurement zone of the cassette 20 when thecassette is inserted into the analyzer 100. The first detector 84 may bepositioned opposite the first light source 82 to detect the amount oflight that passes through the first measurement zone of the cassette 20.As shown illustratively in FIGS. 11 and 12, in one embodiment, theoptical system includes ten light sources and ten detectors. It shouldbe appreciated that in other embodiments, the number of light sourcesand detectors may vary as the invention is not so limited. As mentionedabove, the cassette 20 may include a plurality of measurement zones 209and the cassette 20 may be positioned within the analyzer such that eachmeasurement zone 209 aligns with a light source and correspondingdetector. In some embodiments, the light source includes an opticalaperture 83 (FIG. 11) which may help direct light from the light sourceto a particular region within a measurement zone of the cassette.

In one embodiment, the light sources are light emitting diodes (LED's)or laser diodes. For example, an InGaAlP red semiconductor laser diodeemitting at 654 nm may be used. Other light sources can also be used.The light source, e.g., as shown illustratively in FIG. 14, may bepositioned within a nest or housing 90. The nest or housing 90 mayinclude a narrow aperture or thin tube 92 that may assist in collimatinglight. As shown, the light sources may be positioned above where thecassette 20 is inserted into the analyzer such that the light sourceshines down onto the top surface of the cassette 20. Other suitableconfigurations of the light source with respect to the cassette are alsopossible.

It should be appreciated that the wavelength of the light sources mayvary as the invention is not so limited. For example, in one embodiment,the wavelength of the light source is approximately 670 nm, and inanother embodiment, the wavelength of the light source is approximately650 nm. It should be appreciated that in one embodiment, the wavelengthof each light source may be different such that each measurement zone209 of the cassette receives a different light wavelength. In oneparticular embodiment when measuring hemocrit or hemoglobin, anisobestic wavelength range between approximately 590 nm andapproximately 805 nm may be used for at least one of the measurementzones.

As mentioned, a detector 84 may be spaced apart from and positionedbelow a light source 82 to detect the amount of light that passesthrough the cassette. In one embodiment, one or more of the detectorsare photodetectors (e.g., photodiodes). In certain embodiments, thephotodetector may be any suitable device capable of detecting thetransmission of light that is emitted by the light source. One type ofphotodetector is an optical integrated circuit (IC) including aphotodiode having a peak sensitivity at 700 nm, an amplifier and avoltage regulator. The detector, e.g., as shown in FIG. 14, may bepositioned within a nest or housing 94 which may include a narrowaperture or thin tube 96 to ensure that only light from the center ofthe measurement zone 209 is measured at the detector 84. As described inmore detail below, if the light source is pulse modulated, thephotodetector may include a filter to remove the effect of light that isnot at the selected frequency. When multiple and neighboring signals aredetected at the same time, the light source used for each measurementzone (e.g., detection region) can be modulated at a frequencysufficiently different from that of its neighboring light source. Inthis configuration, the each detector can be configured (e.g., usingsoftware) to select for its attributed light source, thereby avoidinginterfering light form neighboring optical pairs.

As described herein, a cassette may include a measurement zone whichincludes a meandering channel configured and arranged to align with adetector such that upon alignment, the detector can measure a singlesignal through more than one adjacent segment of the meandering channel.In some embodiments, the detector is able to detect a signal within atleast a portion of the area of the meandering channel and through morethan one segment of the meandering channel such that a first portion ofthe signal, measured from a first segment of the meandering channel, issimilar to a second portion of the signal, measured from a secondsegment of the meandering channel. In such embodiments, because thesignal is present as a part of more than one segment of the meanderingchannel, there is no need for precise alignment between a detector and ameasurement zone.

The positioning of the detector over the measurement zone (e.g., ameandering region) without the need for precision is an advantage, sinceexternal (and possibly, expensive) equipment such as microscopes,lenses, and alignment stages are not required (although they may be usedin certain embodiments). Instead, alignment may be performed by low-costmethods that do not necessarily require an active or separate alignmentstep by the user. For example, in one embodiment, a cassette comprisinga meandering region can be placed in a slot of an analyzer describedherein (e.g., in a cavity having the same or similar shape as thecassette), and the measurement zone can be automatically located in abeam of light of the detector. Possible causes of misalignment causedby, for instance, cassette-to-cassette variations, the exact location ofthe cassette in the slot, and normal usage of the cassette, may benegligible compared to the dimensions of the measurement zone. As aresult, the meandering region can stay within the beam of light anddetection is not interrupted due to these variations.

The detector may detect a signal within all, or a portion, of ameasurement zone (e.g., including a meandering region). In other words,different amounts of the meandering region may be used as an opticaldetection pathway. For instance, the detector may detect a signal withinat least 15% of the measurement zone, at least 20% of the measurementzone, at least 25% of the measurement zone, within at least 50% of themeasurement zone, or within at least 75% of the measurement zone (butless than 100% of the measurement zone). The area in which themeasurement zone is used as an optical detection pathway may also dependon, for instance, the opacity of the material in which the channel isfabricated (e.g., whether all, or, a portion, of the channel istransparent), the amount of a non-transparent material that may cover aportion of the channel (e.g., via use of a protective cover), and/or thesize of the detector and the measurement zone.

In one embodiment, a signal produced by a reaction carried out in thecassette is homogenous over the entire measurement zone (e.g., over anentire meandering channel region). That is, the measurement zone (e.g.,meandering channel region) may allow production and/or detection of asingle, homogenous signal in said region upon carrying out a chemicaland/or biological reaction (e.g., and upon detection by a detector).Prior to carrying out a reaction in the meandering channel region, themeandering channel may include, for example, a single species (andconcentration of species) to be detected/determined. The species may beadsorbed to a surface of the meandering channel. In another embodiment,the signal may be homogeneous over only portions of the meanderingregion, and one or more detectors may detect different signals withineach of the portions. In certain instances, more than one measurementzone can be connected in series and each measurement zone can be used todetect/determine a different species. It should be understood that whilemeandering regions are described, measurement zones that do not includemeandering regions can also be used.

Applicant has recognized that the amount of light transmitted through ameasurement zone of the cassette may be used to determine informationabout not only the sample, but also information about specific processesoccurring in the fluidic system of the cassette (e.g., mixing ofreagents, flow rate, etc.). In some cases, measurement of light througha region can be used as feedback to control fluid flow in the system. Incertain embodiments, quality control or abnormalities in the operationof the cassette can be determined. For example, feedback from ameasurement zone to a control system can be used to determineabnormalities that have occurred in the microfluidic system, and thecontrol system may send a signal to one or more components to cause allor portions of the system to shut down. Consequently, the quality of theprocesses being performed in the microfluidic system can be controlledusing the systems and methods described herein. A more detaileddiscussion of such and other processes may be found below and in a U.S.Provisional Patent Application No. 61/325,023, filed Apr. 16, 2010 andentitled “Feedback Control in Microfluidic Systems”, which is hereinincorporated by reference in its entirety.

It should be recognized that a clear liquid (such as water) may allow alarge amount of light to be transmitted from the light source 82,through the measurement zone 209 and to the detector 84. Air within themeasurement zone 209 may lead to less light transmitted through themeasurement zone 209 because more light may scatter within the channelcompared to when a clear liquid is present. When a blood sample is in ameasurement zone 209, a significantly less amount of light may passthrough to the detector 84 due to the light scattering off of bloodcells and also due to absorbance. In one embodiment, silver associateswith a sample component bound to a surface within the measurement zoneand as silver builds up within the measurement zone 209, less and lesslight is transmitted through the measurement zone 209.

It is recognized that measuring the amount of light that is detected ateach detector 84 enables a user to determine which reagents are in aparticular measurement zone 209 at a particular point in time. It isalso recognized that by measuring the amount of light that is detectedwith each detector 84, it is possible to measure the amount of silverdeposited in each measurement zone 209. This amount may correspond tothe amount of analyte captured during a reaction which may thus providea measure of the concentration of the analyte in the sample.

As noted above, Applicant has recognized that the optical system 80 maybe used for a variety of quality control reasons. First, the time ittakes for a sample to reach a measurement zone where the optical systemdetects the light that passes though the measurement zone may be used todetermine whether there is a leak or clog in the system. Also, when thesample is expected to be a certain volume, for example, approximately 10microliters, there is an expected flow time which would be associatedfor the sample to pass through the channels and measurement zones. Ifthe sample falls outside of that expected flow time, it could be anindication that there is not enough sample to conduct the analysisand/or that the wrong type of sample was loaded into the analyzer.Additionally, an expected range of results may be determined based uponthe type of sample (e.g., serum, blood, urine, etc.) and if the sampleis outside of the expected range, it could be an indication of an error.

In one embodiment, the optical system 80 includes a plurality of lightsources 82, 86 and a plurality of corresponding detectors 84, 88. Asshown illustratively in FIGS. 11-13, in one embodiment, a first lightsource 82 is adjacent a second light source 86, where the first lightsource 82 is configured to pass light though a first measurement zone ofthe cassette 20 and the second light source is configured to pass lightthrough a second measurement zone of the cassette 20. In one embodiment,the light sources are configured such that the second light source 86 isnot activated unless the first light source 82 is deactivated. Applicanthas recognized that some light from one light source may spread over toan adjacent detector and may affect the amount of light detected at theadjacent detector. In one set of embodiments, if the adjacent lightsource is activated at the same time as the first light source, thenboth detectors 84, 88 are also measuring the amount of light that passesthrough the first and second measurement zones of the cassette at thesame time, which may lead to inaccurate measurements.

Thus, in one set of embodiments, the plurality of light sources areconfigured to activate sequentially with only one light source activatedat a time. The corresponding detector for the activated light source isthus only detecting the amount of light that passes through thecorresponding measurement zone 209. In one particular embodiment, thelight sources are configured to each activate for a short period of time(e.g., at least approximately 500, 250, 100, or 50 microseconds, or, insome embodiments, less than or equal to approximately 500, 250, 100, or50 microseconds), and then an adjacent light source is configured toactivate for a similar time frame. Activation for 100 microsecondscorresponds to a rate of 10 kHz. In one embodiment, a multiplexed analogto digital converter is used to pulse the light and measure the amountof light detected at each corresponding detector every 500, 250, 100, or50 microseconds. Pulsing the light in this manner may help to preventstray light passing through one measurement zone to alter the amount oflight detected that passes through an adjacent measurement zone.

Although there may be some benefits associated with pulsing the lightsources in the manner described above, it should be recognized that theinvention is not so limited and that other arrangements may be possible,such as where multiple light sources may be activated at the same time.For example, in one embodiment, light sources that are not directlyadjacent to one another can be activated substantially simultaneously.

Referring to FIG. 17, in one embodiment, the analyzer 100 includes atemperature regulating system positioned within the housing 101 whichmay be configured to regulate the temperature within the analyzer. Forcertain sample analysis, the sample may need to be kept within a certaintemperature range. For example, in one embodiment, it is desirable tomaintain the temperature within the analyzer 100 at approximately 37° C.Accordingly, in one embodiment, the temperature regulating systemincludes a heater 140 configured to heat the cassette 20. In oneembodiment, the heater 140 is a resistive heater which may be positionedon the underside of where the cassette 20 is placed in the analyzer 100.In one embodiment, the temperature regulating system also includes athermistor 142 to measure the temperature of the cassette 20 and acontroller circuit may be provided to control the temperature.

In one embodiment, the passive flow of air within the analyzer may actto cool the air within the analyzer if needed. A fan (not shown) mayoptionally be provided in the analyzer 100 to lower the temperaturewithin the analyzer 100. In some embodiments, the temperature regulatingsystem may include Peltier thermoelectric heaters and/or coolers withinthe analyzer.

In certain embodiments, an identification system including one or moreidentifiers is used and associated with one or more components ormaterials associated with a cassette and/or analyzer. The “identifiers,”as described in greater detail below, may themselves be “encoded with”information (i.e. carry or contain information, such as by use of aninformation carrying, storing, generating, or conveying device such as aradio frequency identification (RFID) tag or bar code) about thecomponent including the identifier, or may not themselves be encodedwith information about the component, but rather may only be associatedwith information that may be contained in, for example, a database on acomputer or on a computer readable medium (e.g., information about auser, and/or sample to be analyzed). In the latter instance, detectionof such an identifier can trigger retrieval and usage of the associatedinformation from the database.

Identifiers “encoded with” information about a component need notnecessarily be encoded with a complete set of information about thecomponent. For example, in certain embodiments, an identifier may beencoded with information merely sufficient to enable a uniqueidentification of the cassette (e.g. relating to a serial no., part no.,etc.), while additional information relating to the cassette (e.g. type,use (e.g., type of assay), ownership, location, position, connectivity,contents, etc.) may be stored remotely and be only associated with theidentifier.

“Information about” or “information associated with” a cassette,material, or component, etc. is information regarding the identity,positioning, or location of the cassette, material or component or theidentity, positioning, or location of the contents of a cassette,material or component and may additionally include information regardingthe nature, state or composition of the cassette, material, component orcontents. “Information about” or “information associated with” acassette, material or component or its contents can include informationidentifying the cassette, material or component or its contents anddistinguishing the cassette, material, component or its contents fromothers. For example, “information about” or “information associatedwith” a cassette, material or component or its contents may refer toinformation indicating the type or what the cassette, material orcomponent or its contents is, where it is or should be located, how itis or should be positioned, the function or purpose of the cassette,material or component or its contents, how the cassette, material orcomponent or its contents is to be connected with other components ofthe system, the lot number, origin, calibration information, expirationdate, destination, manufacturer or ownership of the cassette, materialor component or its contents, the type of analysis/assay to be performedin the cassette, information about whether the cassette has beenused/analyzed, etc.

In one set of embodiments, an identifier is associated with a cassetteand/or analyzer described herein. In general, as used herein, the term“identifier” refers to an item capable of providing information aboutthe cassette and/or analyzer (e.g. information including one or more ofidentity, location, or position/positioning of the cassette and/oranalyzer or a component thereof) with which the identifier is associatedor installed into, or capable of being identified or detected and theidentification or detection event being associated with informationabout the cassette and/or analyzer with which the identifier isassociated. Non-limiting examples of identifiers that may be used in thecontext of the invention include radio frequency identification (RFID)tags, bar codes, serial numbers, color tags, fluorescent or optical tags(e.g., using quantum dots), chemical compounds, radio tags, magnetictags, among others.

In one embodiment, as shown illustratively in FIG. 16, the analyzer 100includes an identification reader 60 positioned within the housing 101configured to read information about with the cassette 20. Any suitableidentification reader that can be used to read information from anidentifier. Non-limiting examples of identification readers include RFIDreaders, bar code scanners, chemical detectors, cameras, radiationdetectors, magnetic or electric field detectors, among others. Themethod of detection/reading and appropriate type of identificationdetector depends on the particular identifier utilized and can include,for example, optical imaging, fluorescence excitation and detection,mass spectrometry, nuclear magnetic resonance, sequencing,hybridization, electrophoresis, spectroscopy, microscopy, etc. In someembodiments, the identification readers may be mounted or pre-embeddedin specific locations (e.g., on or within a cassette and/or analyzer).

In one embodiment, the identification reader 60 is an RFID readerconfigured to read an RFID identifier associated with the cassette 20.For example, as shown illustratively in FIG. 2, in one embodiment, theanalyzer 100 includes an RFID module and antenna that are configured toread information from the cassette 20 inserted into the analyzer 100. Inanother embodiment, the identification reader 60 is a barcode readerconfigured to read a barcode associated with the cassette 20. Once thecassette 20 is inserted into the analyzer 100, the identification reader60 may read the information from the cassette 20. The identifier on thecassette may include one or more of the types of information such ascassette type, type of analysis/assay to be performed, lot number,information about whether the cassette has been used/analyzed, and otherinformation described herein. The reader 60 may also be configured toread information provided with a group of cassettes, such as in a box ofcassettes, such as, but not limited to calibration information,expiration date, and any additional information specific to that lot.The information identified may be optionally displayed to a user, e.g.,to confirm that a correct cassette and/or type of assay is beingperformed.

In some cases, the identification reader may be integrated with acontrol system via communication pathways. Communication between theidentification readers and the control system may occur along ahard-wired network or may be transmitted wirelessly. In one embodiment,the control system can be programmed to recognize a specific identifier(e.g., of a cassette associated with information relating to a cassettetype, manufacturer, assay to be performed, etc.) as indicating thecassette is suitably connected or inserted within a particular type ofanalyzer.

In one embodiment, the identifier of a cassette be associated withpredetermined or programmed information contained in a databaseregarding the use of the system or cassette for a particular purpose,user or product, or with particular reaction conditions, sample types,reagents, users, and the like. If an incorrect match is detected or anidentifier has been deactivated, the process may be halted or the systemmay be rendered not operable until the user has been notified, or uponacknowledgement by a user.

The information from or associated with an identifier can, in someembodiments, be stored, for example in computer memory or on a computerreadable medium, for future reference and record-keeping purposes. Forexample, certain control systems may employ information from orassociated with identifiers to identify which components (e.g.,cassettes) or type of cassettes were used in a particular analysis, thedate, time, and duration of use, the conditions of use, etc. Suchinformation may be used, for example, to determine whether one or morecomponents of the analyzer should be cleaned or replaced. Optionally, acontrol system or any other suitable system could generate a report fromgathered information, including information encoded by or associatedwith the identifiers, that may be used in providing proof of compliancewith regulatory standards or verification of quality control.

Information encoded on or associated with an identifier may also beused, for example, to determine whether the component associated withthe identifier (e.g., a cassette) is authentic or counterfeit. In someembodiments, the determination of the presence of a counterfeitcomponent causes system lockout. In one example, the identifier maycontain a unique identity code. In this example, the process controlsoftware or analyzer would not permit system startup (e.g., the systemmay be disabled) if a foreign or mismatched identity code (or noidentity code) was detected.

In certain embodiments, the information obtained from or associated withan identifier can be used to verify the identity of a customer to whomthe cassette and/or analyzer is sold or for whom a biological, chemical,or pharmaceutical process is to be performed. In some cases, theinformation obtained from or associated with an identifier is used aspart of a process of gathering data for troubleshooting a system. Theidentifier may also contain or be associated with information such asbatch histories, assembly process and instrumentation diagrams (P andIDs), troubleshooting histories, among others. Troubleshooting a systemmay be accomplished, in some cases, via remote access or include the useof diagnostic software.

In one embodiment, the analyzer 100 includes a user interface 200, whichmay be positioned within the housing 101 and configured for a user toinput information into the sample analyzer 100. In one embodiment, theuser interface 200 is a touch screen, which is illustrated in FIG. 1 andFIGS. 16-21.

As set forth in FIGS. 16-21, the touch screen may guide a user throughthe operation of the analyzer 100, providing text and/or graphicalinstructions for use of the analyzer 100. FIG. 17 illustrates oneexample of the graphics for the touch screen user interface 200 at thebeginning of the sample analysis process. FIG. 18 illustrates oneexample of the graphics for the touch screen user interface 200 whichguides the user to insert the cassette 20 into the analyzer 100. FIG. 19illustrates one example of the graphics for the touch screen userinterface 200 which guides the user to input the patient's name or otherpatient identification source/number into the analyzer 100. It should beappreciated that the patient information such as name, date of birth,and/or patient ID number may be inputted into the touch screen userinterface to identify the patient. FIG. 20 illustrates one example ofthe graphics for the touch screen user interface 200 while the sample isbeing analyzed. As shown, the touch screen may indicate the amount oftime remaining to complete the analysis of the sample. Finally, FIG. 21illustrates one example of the graphics for the touch screen userinterface 200 which illustrates the results of the sample analysis alongwith the patient's name or other identifying information.

In another embodiment, the user interface may be configured differently,such as with an LCD display and a single button scroll through menu. Inanother embodiment, the user interface may simply include a start buttonto activate the analyzer. In other embodiments, the user interface fromseparate independent devices (such as a smart phone or mobile computer)can be used to interface with the analyzer.

The above-described analyzer 100 may be used in a variety of ways toprocess and analyze a sample placed within the analyzer. In oneparticular embodiment, once the mechanical component 121 configured tointerface with the cassette indicates that the cassette 20 is properlyloaded in the analyzer 100, the identification reader 60 reads andidentifies information associated with the cassette 20. The analyzer 100may be configured to compare the information to data stored in a controlsystem to ensure that it has calibration information for this particularsample. In the event that the analyzer does not have the propercalibration information, the analyzer may output a request to the userto upload the specific information needed. The analyzer may also beconfigured to review expiration date information associated with thecassette and cancel the analysis if the expiration date has passed.

In one embodiment, once the analyzer 100 has determined that thecassette 20 may be analyzed, a fluid flow source such as the vacuummanifold 48 may be configured to contact the cassette 20 to ensure anairtight seal around the vacuum port 219 and vent ports 215. In oneembodiment, the optical system 80 may take initial measurements toobtain reference readings. Such reference readings may be taken bothwith the light sources 82, 86 activated and deactivated.

To initiate movement of the sample, the vacuum system 40 may beactivated, which may rapidly change the pressure within the channel 206,207 (e.g., reduced to approximately −30 kPa). This reduction of pressurewithin the channel may drive the sample into the channel 206 and througheach of the measurement zones 209A-209D (see FIG. 8). After the samplereaches the final measurement zone 209D, the sample may continue to flowinto the liquid containment region 217.

In one particular embodiment, the microfluidic sample analyzer 100 isused to measure the level of a prostate specific antigen (PSA) in ablood sample. In this embodiment, four measurement zones 209A-209D maybe utilized to analyze the sample. For example, in a first measurementzone, the walls of the channel may be blocked with a blocking protein(such as Bovine Serum Albumin) such that little or no proteins in theblood sample attach to the walls of the measurement zone 209 (except forperhaps some non-specific binding which may be washed off). This firstmeasurement zone may act as a negative control.

In a second measurement zone 209, the walls of the channel 206 may becoated with a predetermined large quantity of a prostate specificantigen (PSA) to act as a high or positive control. As the blood samplepasses through the second measurement zone 209, little or no PSAproteins in the blood may bind to the walls of the channel. Goldconjugated signal antibodies in the sample may be dissolved from insideof the fluidic connector tube 222 or may be flowed from any othersuitable location. These antibodies may not yet be bound to the PSA inthe sample, and thus they may bind to the PSA on the walls of thechannel to act as a high or positive control.

In a third measurement zone 209, the walls of the channel 206 may becoated with a predetermined small quantity of PSA to act as a lowcontrol. As the blood sample flows through this measurement zone 209, noPSA proteins in the sample bind to the wall of the channel. Goldconjugated signal antibodies in the sample may be dissolved from insideof the fluidic connector tube 222 (which are not yet bound to the PSA inthe sample) or may be flowed from any other suitable location, and maybind to the PSA on the walls of the channel to act as a low control.

In a fourth measurement zone 209, the walls of the channel 206 may becoated with the capture antibody, an anti-PSA antibody, which binds to adifferent epitope on the PSA protein than the gold conjugated signalantibody. As the blood sample flows through the fourth measurement zone,PSA proteins in the blood sample may bind to the anti-PSA antibody in away that is proportional to the concentration of these proteins in theblood. Thus, in one embodiment, the first three measurement zones 209may act as controls and the fourth measurement zone 209 may actuallytest the sample. In other embodiments, different numbers of measurementzones can be provided, and an analysis may optionally include more thanone measurement zones that actually test the sample.

In some instances, measurements from a region that analyzes the sample(e.g., the fourth measurement zone described above) can be used not onlyto determine the concentration of an analyte in a sample, but also as acontrol as well. For example, a threshold measurement can be establishedat an early phase of amplification. Measurements above this value (orbelow this value) may indicate that the concentration of analyte isoutside the desired range for the assay. This technique may be used toidentify, for example, whether a High Dose Hook Effect is taking placeduring the analysis, i.e., when a very high concentration of analytegives an artificially low reading.

In other embodiments, different numbers of measurement zones can beprovided, and an analysis may optionally include more than onemeasurement zones that actually test the sample. Additional measurementzones can be used to measure additional analytes so that the system canperform multiplex assays simultaneously with a single sample.

In one particular embodiment, it takes approximately eight minutes for a10 microliter blood sample to flow through the four measurement zones209. The start of this analysis may be calculated when the pressurewithin the channel 206 is approximately −30 kPa. During this time, theoptical system 80 is measuring the light transmission for eachmeasurement zone, and in one embodiment, this data may be transmitted toa control system approximately every 0.1 seconds. Using referencevalues, these measurements may be converted using the followingformulas:

Transmission=(l−ld)/(lr−ld)  (1)

Where:

-   -   l=the intensity of transmitted light through a measurement zone        at a given point in time    -   ld=the intensity of transmitted light through a measurement zone        with the light source off    -   lr=a reference intensity (i.e. the intensity of the transmitted        light at a measurement zone with the light source activated, or        before the start of an analysis when only air is in the channel        and

Optical Density=−log(Transmission)  (2)

Thus, using these formulas, the optical density in a measurement zone209 may be calculated.

As described herein, a variety of methods can be used to control fluidflow in a cassette, including the use of pumps, vacuums, valves, andother components associated with an analyzer. In some cases, fluidcontrol can also be performed at least in part by one or more componentswithin the cassette, such as by using a valve positioned within thecassette, or the use of specific fluids and channel configurations withthe cassette. In one set of embodiments, control of fluid flow can beachieved based at least in part on the influence of channel geometry andthe viscosity of one or more fluids (which may be stored) inside thecassette.

One method includes flowing a plug of a low viscosity fluid and a plugof a high viscosity fluid in a channel including a flow constrictionregion and a non-constriction region. In one embodiment, the lowviscosity fluid flows at a first flow rate in the channel and the flowrate is not substantially affected by the fluid flowing in the flowconstriction region. When the high viscosity fluid flows from thenon-constriction region to the flow constriction region, the flow ratesof the fluids decrease substantially, since the flow rates, in somesystems, are influenced by the highest viscosity fluid flowing in thesmallest cross-sectional area of the system (e.g., the flow constrictionregion). This causes the low viscosity fluid to flow at a second, slowerflow rate than its original flow rate, e.g., at the same flow rate atwhich the high viscosity fluid flows in the flow constriction region.

For example, one method of controlling fluid flow may involve flowing afirst fluid from a first channel portion to a second channel portion ina microfluidic system, wherein a fluid path defined by the first channelportion has a larger cross-sectional area than a cross-sectional area ofa fluid path defined by the second channel portion, and flowing a secondfluid in a third channel portion in the microfluidic system in fluidcommunication with the first and second channel portions, wherein theviscosity of the first fluid is different than the viscosity of thesecond fluid, and wherein the first and second fluids are substantiallyincompressible. Without stopping the first or second fluids, avolumetric flow rate of the first and second fluids may be decreased bya factor of at least 3, at least 10, at least 20, at least 30, at least40, or at least 50 in the microfluidic system as a result of the firstfluid flowing from the first channel portion to the second channelportion, compared to the absence of flowing the first fluid from thefirst channel portion to the second channel portion. A chemical and/orbiological interaction involving a component of the first or secondfluids may take place at a first measurement zone in fluid communicationwith the channel portions while the first and second fluids are flowingat the decreased flow rate.

Accordingly, by designing microfluidic systems with flow constrictionregions positioned at particular locations and by choosing appropriateviscosities of fluids, a fluid can be made to speed up or slow down atdifferent locations within the system without the use of valves and/orwithout external control. In addition, the length of the channelportions can be chosen to allow a fluid to remain in a particular areaof the system for a certain period of time. Such systems areparticularly useful for performing chemical and/or biological assays, aswell as other applications in which timing of reagents is important.Non-limiting examples of methods and configurations of channels forcontrolling fluid flow are described in more detail in U.S. patentapplication Ser. No. 12/428,372, filed Apr. 22, 2009, published as U.S.Patent Publication No. 2009/0266421, entitled “Flow Control inMicrofluidic Systems”, which is incorporated herein by reference in itsentirety for all purposes.

FIG. 16 is a block diagram 300 that illustrates how a control system 305(see FIG. 13) may be operatively associated with a variety of differentcomponents according to one embodiment. Control systems described hereincan be implemented in numerous ways, such as with dedicated hardware orfirmware, using a processor that is programmed using microcode orsoftware to perform the functions recited above or any suitablecombination of the foregoing. A control system may control one or moreoperations of a single analysis (e.g., for a biological, biochemical orchemical reaction), or of multiple (separate or interconnected)analyses. As shown illustratively in FIG. 13, the control system 305 maybe positioned within the housing 101 of the analyzer and may beconfigured to communicate with the identification reader 60, the userinterface 200, the fluid flow source 40, the optical system 80, and/orthe temperature regulating system to analyze a sample in the cassette.

In one embodiment, the control system includes at least two processors,including a real time processor that controls and monitors all of thesub-systems which directly interface with the cassette. In oneembodiment, at a particular time interval (e.g., every 0.1 seconds),this processor communicates with a second higher level processor whichcommunicates with the user through the user interface and/or thecommunication sub-system (discussed below) and directs the operation ofthe analyzer (e.g., determines when to start analyzing a sample andinterprets the results). In one embodiment, communication between thesetwo processors occurs through a serial communication bus. It should beappreciated that in another embodiment, the analyzer may only includeone processor, or more than two processors, as the invention is not solimited.

In one embodiment, the analyzer is capable of interfacing with externaldevices and may, for example, include ports for connection with one ormore external communication units. External communication may beaccomplished, for example, via USB communication. For example, as shownin FIG. 16, the analyzer may output the results of a sample analysis toa USB printer 400, or to a computer 402. Additionally, the data streamproduced by the real time processor may be outputted to a computer or aUSB memory stick 404. In some embodiments, a computer may be able todirectly control the analyzer through a USB connection as well. Further,other types of communication options are available as the presentinvention is not limited in this respect. For example, Ethernet,Bluetooth and/or WI-FI communication 406 with the analyzer may beestablished through the processor.

The calculation methods, steps, simulations, algorithms, systems, andsystem elements described herein may be implemented using a computerimplemented control system, such as the various embodiments of computerimplemented systems described below. The methods, steps, systems, andsystem elements described herein are not limited in their implementationto any specific computer system described herein, as many otherdifferent machines may be used.

The computer implemented control system can be part of or coupled inoperative association with a sample analyzer, and, in some embodiments,configured and/or programmed to control and adjust operationalparameters of the sample analyzer, as well as analyze and calculatevalues, as described above. In some embodiments, the computerimplemented control system can send and receive reference signals to setand/or control operating parameters of the sample analyzer and,optionally, other system apparatus. In other embodiments, the computerimplemented system can be separate from and/or remotely located withrespect to the sample analyzer and may be configured to receive datafrom one or more remote sample analyzer apparatus via indirect and/orportable means, such as via portable electronic data storage devices,such as magnetic disks, or via communication over a computer network,such as the Internet or a local intranet.

The computer implemented control system may include several knowncomponents and circuitry, including a processing unit (i.e., processor),a memory system, input and output devices and interfaces (e.g., aninterconnection mechanism), as well as other components, such astransport circuitry (e.g., one or more busses), a video and audio datainput/output (I/O) subsystem, special-purpose hardware, as well as othercomponents and circuitry, as described below in more detail. Further,the computer system may be a multi-processor computer system or mayinclude multiple computers connected over a computer network.

The computer implemented control system may include a processor, forexample, a commercially available processor such as one of the seriesx86, Celeron and Pentium processors, available from Intel, similardevices from AMD and Cyrix, the 680X0 series microprocessors availablefrom Motorola, and the PowerPC microprocessor from IBM. Many otherprocessors are available, and the computer system is not limited to aparticular processor.

A processor typically executes a program called an operating system, ofwhich WindowsNT, Windows95 or 98, UNIX, Linux, DOS, VMS, MacOS and OS8are examples, which controls the execution of other computer programsand provides scheduling, debugging, input/output control, accounting,compilation, storage assignment, data management and memory management,communication control and related services. The processor and operatingsystem together define a computer platform for which applicationprograms in high-level programming languages are written. The computerimplemented control system is not limited to a particular computerplatform.

The computer implemented control system may include a memory system,which typically includes a computer readable and writeable non-volatilerecording medium, of which a magnetic disk, optical disk, a flash memoryand tape are examples. Such a recording medium may be removable, forexample, a floppy disk, read/write CD or memory stick, or may bepermanent, for example, a hard drive.

Such a recording medium stores signals, typically in binary form (i.e.,a form interpreted as a sequence of one and zeros). A disk (e.g.,magnetic or optical) has a number of tracks, on which such signals maybe stored, typically in binary form, i.e., a form interpreted as asequence of ones and zeros. Such signals may define a software program,e.g., an application program, to be executed by the microprocessor, orinformation to be processed by the application program.

The memory system of the computer implemented control system also mayinclude an integrated circuit memory element, which typically is avolatile, random access memory such as a dynamic random access memory(DRAM) or static memory (SRAM). Typically, in operation, the processorcauses programs and data to be read from the non-volatile recordingmedium into the integrated circuit memory element, which typicallyallows for faster access to the program instructions and data by theprocessor than does the non-volatile recording medium.

The processor generally manipulates the data within the integratedcircuit memory element in accordance with the program instructions andthen copies the manipulated data to the non-volatile recording mediumafter processing is completed. A variety of mechanisms are known formanaging data movement between the non-volatile recording medium and theintegrated circuit memory element, and the computer implemented controlsystem that implements the methods, steps, systems and system elementsdescribed above in relation to FIG. 16 is not limited thereto. Thecomputer implemented control system is not limited to a particularmemory system.

At least part of such a memory system described above may be used tostore one or more data structures (e.g., look-up tables) or equationsdescribed above. For example, at least part of the non-volatilerecording medium may store at least part of a database that includes oneor more of such data structures. Such a database may be any of a varietyof types of databases, for example, a file system including one or moreflat-file data structures where data is organized into data unitsseparated by delimiters, a relational database where data is organizedinto data units stored in tables, an object-oriented database where datais organized into data units stored as objects, another type ofdatabase, or any combination thereof.

The computer implemented control system may include a video and audiodata I/O subsystem. An audio portion of the subsystem may include ananalog-to-digital (A/D) converter, which receives analog audioinformation and converts it to digital information. The digitalinformation may be compressed using known compression systems forstorage on the hard disk to use at another time. A typical video portionof the I/O subsystem may include a video image compressor/decompressorof which many are known in the art. Such compressor/decompressorsconvert analog video information into compressed digital information,and vice-versa. The compressed digital information may be stored on harddisk for use at a later time.

The computer implemented control system may include one or more outputdevices. Example output devices include a cathode ray tube (CRT)display, liquid crystal displays (LCD) and other video output devices,printers, communication devices such as a modem or network interface,storage devices such as disk or tape, and audio output devices such as aspeaker.

The computer implemented control system also may include one or moreinput devices. Example input devices include a keyboard, keypad, trackball, mouse, pen and tablet, communication devices such as describedabove, and data input devices such as audio and video capture devicesand sensors. The computer implemented control system is not limited tothe particular input or output devices described herein.

It should be appreciated that one or more of any type of computerimplemented control system may be used to implement various embodimentsdescribed herein. Aspects of the invention may be implemented insoftware, hardware or firmware, or any combination thereof. The computerimplemented control system may include specially programmed, specialpurpose hardware, for example, an application-specific integratedcircuit (ASIC). Such special-purpose hardware may be configured toimplement one or more of the methods, steps, simulations, algorithms,systems, and system elements described above as part of the computerimplemented control system described above or as an independentcomponent.

The computer implemented control system and components thereof may beprogrammable using any of a variety of one or more suitable computerprogramming languages. Such languages may include procedural programminglanguages, for example, C, Pascal, Fortran and BASIC, object-orientedlanguages, for example, C++, Java and Eiffel and other languages, suchas a scripting language or even assembly language.

The methods, steps, simulations, algorithms, systems, and systemelements may be implemented using any of a variety of suitableprogramming languages, including procedural programming languages,object-oriented programming languages, other languages and combinationsthereof, which may be executed by such a computer system. Such methods,steps, simulations, algorithms, systems, and system elements can beimplemented as separate modules of a computer program, or can beimplemented individually as separate computer programs. Such modules andprograms can be executed on separate computers.

Such methods, steps, simulations, algorithms, systems, and systemelements, either individually or in combination, may be implemented as acomputer program product tangibly embodied as computer-readable signalson a computer-readable medium, for example, a non-volatile recordingmedium, an integrated circuit memory element, or a combination thereof.For each such method, step, simulation, algorithm, system, or systemelement, such a computer program product may comprise computer-readablesignals tangibly embodied on the computer-readable medium that defineinstructions, for example, as part of one or more programs, that, as aresult of being executed by a computer, instruct the computer to performthe method, step, simulation, algorithm, system, or system element.

It should be appreciated that various embodiments may be formed with oneor more of the above-described features. The above aspects and featuresmay be employed in any suitable combination as the present invention isnot limited in this respect. It should also be appreciated that thedrawings illustrate various components and features which may beincorporated into various embodiments. For simplification, some of thedrawings may illustrate more than one optional feature or component.However, the invention is not limited to the specific embodimentsdisclosed in the drawings. It should be recognized that the inventionencompasses embodiments which may include only a portion of thecomponents illustrated in any one drawing figure, and/or may alsoencompass embodiments combining components illustrated in multipledifferent drawing figures.

EXAMPLES

The following example is intended to illustrate certain features of thepresent invention, but do not exemplify the full scope of the invention.

Example 1

This example describes the use of a cassette and analyzer to perform anassay to detect PSA in a sample by electrolessly depositing silver ontogold particles that are associated with the sample. FIG. 22 includes aschematic illustration of a microfluidic system 500 of a cassette usedin this example. The cassette had a similar shape to cassette 20 shownin FIG. 3. The microfluidic system used in this example is generallydescribed in International Patent Publication No. WO2005/066613(International Patent Application Serial No. PCT/US2004/043585), filedDec. 20, 2004 and entitled “Assay Device and Method,” which isincorporated herein by reference in its entirety for all purposes.

The microfluidic system included measurement zones 510A-510D, wastecontainment region 512, and an outlet 514. The measurement zonesincluded a microfluidic channel 50 microns deep and 120 microns wide,with a total length of 175 mm. The microfluidic system also includedmicrofluidic channel 516 and channel branches 518 and 520 (with inlets519 and 521, respectively). Channel branches 518 and 520 were 350microns deep and 500 microns wide. Channel 516 was formed ofsub-channels 515, which were 350 microns deep and 500 microns widelocated on alternating sides of the cassette, connected by through holes517 having a diameter of approximately 500 microns. Although FIG. 22shows that reagents were stored on a single side of the cassette, inother embodiments, reagents were stored on both sides of the cassette.Channel 516 had a total length of 390 mm, and branches 518 and 520 wereeach 360 mm long. Before sealing the channels, anti-PSA antibodies wereattached to a surface of the microfluidic system in a segment of themeasurement zone 510.

Prior to first use, the microfluidic system was loaded with liquidreagents which were stored in the cassette. A series of 7 wash plugs523-529 (either water of buffer, approximately 2 microliters each) wereloaded using a pipette into sub-channels 515 of channel 516 using thethru-holes. Each of the wash plugs was separated by plugs of air. Fluid528, containing a solution of silver salt, was loaded into branchingchannel through port 519 using a pipette. Fluid 530, containing areducing solution, was loaded into branching channel 520 through port521. Each of the liquids shown in FIG. 9 were separated from the otherliquids by plugs of air. Ports 514, 519, 521, 536, 539, and 540 weresealed with an adhesive tape that can be easily removed or pierced. Assuch, the liquids were stored in the microfluidic system prior to firstuse.

At first use, the ports 514, 519, 521, 536, 539, and 540 were unsealedby a user peeling off a tape covering the opening of the ports. A tube544 containing lyophilized anti-PSA antibodies labeled with colloidalgold and to which 10 microliters of sample blood (522) was added, wasconnected to ports 539 and 540. The tube was part of a fluid connectorhaving a shape and configuration shown in FIG. 3. This created a fluidicconnection between measurement zone 510 and channel 516, which wereotherwise unconnected and not in fluid communication with one anotherprior to first use.

The cassette including microfluidic system 500 was inserted into anopening of an analyzer (e.g., as shown in FIGS. 10, 12 and 17). Thehousing of the analyzer included an arm positioned within the housingthat was configured to engage a cammed surface on the cassette. The armextended at least partially into the opening in the housing such that asthe cassette was inserted into the opening, the arm was pushed away fromthe opening into a second position allowing the cassette to enter theopening. Once the arm engaged the inwardly cammed surface of thecassette, the cassette was positioned and retained within the housing ofthe analyzer, and the bias of the spring prevented the cassette fromslipping out of the analyzer. The analyzer senses the cassette'sinsertion by means of a position sensor.

An identification reader (RFID reader) positioned within the housing ofthe analyzer was used to read an RFID tag on the cassette which includeslot identification information. The analyzer used this identifier tomatch lot information (e.g., calibration information, expiration date ofthe cassette, verification that the cassette is new, and the type ofanalysis/assay to be performed in the cassette) stored in the analyzer.The user was prompted to input information about the patient (from whichthe sample was acquired) into the analyzer using the touch screen. Afterthe information about the cassette was verified by the user, the controlsystem initiated the analysis.

The control system included programmed instructions to perform theanalysis. To initiate the analysis, a signal was sent to the electronicscontrolling a vacuum system, which was a part of the analyzer and usedto provide fluid flow. A manifold with o-rings was pressed against thecassette surface by a solenoid. One port on the manifold sealed (by ano-ring) to port 536 of the microfluidic system of the cassette. Thisport on the manifold was connected by a tube to a simple solenoid valve(SMC V124A-6G-M5, not shown) which was open to the atmosphere. Aseparate vacuum port on the manifold sealed (by-o-ring) to port 514 ofthe microfluidic system of the cassette. A vacuum of approximately −30kPa was applied to port 514. Throughout the analysis, the channelincluding measurement zone 510 positioned between ports 540 and 514 hada substantially constant non-zero pressure drop of approximately −30kPa. Sample 522 was flowed in the direction of arrow 538 into each ofmeasurement zones 510A-510D. As the fluid passed through the measurementzones, the PSA proteins in sample 522 were captured by anti-PSAantibodies immobilized on the measurement zone walls, as described inmore detail below. The sample took about 7-8 minutes to pass through themeasurement zone, after which it was captured in the waste containmentregion 512.

Initiation of the analysis also involved the control system sending asignal to the optical detectors, which were positioned adjacent each ofmeasurement zones 510, to initiate detection. Each of the detectorsassociated with the measurement zones recorded the transmission of lightthrough the channels of the measurement zones, as shown in a plot 600illustrated in FIG. 10. As the sample passed by each of the measurementzones, peaks 610A-610D were produced. The peaks (and troughs) measuredby the detectors are signals (or are converted to signals) that are sentto the control system which compared the measured signals to referencesignals or values pre-programmed into the control system. The controlsystem included a pre-programmed set of instructions for providingfeedback to the microfluidic system based at least in part on thecomparison of signals/values.

In a first measurement zone 510-A of device 500 of FIG. 22, the walls ofthe channel of this measurement zone were blocked with a blockingprotein (Bovine Serum Albumin) prior to first use (e.g., prior tosealing the device). Little or no proteins in the blood sample attachedto the walls of the measurement zone 510-A (except for perhaps somenon-specific binding which may be washed off). This first measurementzone acted as a negative control.

In a second measurement zone 510-B, the walls of the channel of thismeasurement zone were coated with a predetermined large quantity of aprostate specific antigen (PSA) prior to first use (e.g., prior tosealing the device) to act as a high or positive control. As the bloodsample passed through the second measurement zone 510-B, little or noPSA proteins in the blood bound to the walls of the channel. Goldconjugated signal antibodies in the sample may not yet be bound to thePSA in the sample, and thus they may bind to the PSA on the walls of thechannel to act as a high or positive control.

In a third measurement zone 510-C, the walls of the channel of thismeasurement zone were coated with a predetermined low quantity of PSAprior to first use (e.g., prior to sealing the device) to act as a lowcontrol. As the blood sample flowed through this measurement zone,little or no PSA proteins in the sample bind to the wall of the channel.Gold conjugated signal antibodies in the sample may bind to the PSA onthe walls of the channel to act as a low control.

In a fourth measurement zone 510-D, the walls of the channel of thismeasurement zone were coated with the capture antibody, an anti-PSAantibody, which binds to a different epitope on the PSA protein than thegold conjugated signal antibody. The walls were coated prior to firstuse (e.g., prior to sealing the device). As the blood sample flowedthrough the fourth measurement zone during use, PSA proteins in theblood sample bound to the anti-PSA antibody in a way that isproportional to the concentration of these proteins in the blood. Sincethe sample, which included PSA, also included gold-labeled anti-PSAantibodies coupled to the PSA, the PSA captured on the measurement zonewalls formed a sandwich immunocomplex.

Wash fluids 523-529 followed the sample through the measurement zones510 towards waste containment region 512 in the direction of arrow 538.As the wash fluids were passed through the measurement zones, theywashed away remaining unbound sample components. Each wash plug cleanedthe channels of the measurement zones, providing progressively morecomplete cleaning. The last wash fluid 529 (water) washed away saltsthat could react with silver salts (e.g., chloride, phosphate, azide).

As shown in the plot illustrated in FIG. 23, while the wash fluids wereflowing through the measurement zones, each of the detectors associatedwith the measurement zones measures a pattern 620 of peaks and troughs.The troughs corresponded to the wash plugs (which are clear liquids andthus provide maximum light transmission). The peaks between each plugrepresent the air between each plug of clear liquid. Since the assayincluded 7 wash plugs, 7 troughs and 7 peaks are present in plot 600.The first trough 622 is generally not as deep as the other troughs 624since the first wash plug often catches blood cells left in the channeland thus is not completely clear.

The final peak of air 628 is much longer than the previous peaks becausethere were no wash plugs to follow. As a detector detects the length ofthis air peak, one or more signals is sent to the control system whichcompares the length of time of this peak to a pre-set reference signalor input value having a particular length. If the length of time of themeasured peak is long enough compared to the reference signal, thecontrol system sends a signal to the electronics controlling vent valve536 to actuate the valve and initiate mixing of fluids 528 and 530.(Note that the signal of peak of air 628 may be combined with a signalindicating either 1) the intensity of the peak; 2) where this peak ispositioned as a function of time, and/or 3) one or more signalsindicating that a series of peaks 620 of particular intensity hasalready passed. In this way, the control system distinguishes peak ofair 628 from other peaks of long duration such as peak 610 from thesample, e.g., using a pattern of signals.)

To initiate mixing, the solenoid connected by the manifold to vent port536 is closed. Since the vacuum remains on and no air can enter throughvent valve 536, air enters the device through ports 519 and 521 (whichare open). This forces the two fluids 528 and 530 in the two storagechannels upstream of vent valve 536 to move substantially simultaneouslytoward outlet 514. These reagents mix at the intersection of thechannels to form an amplification reagent (a reactive silver solution)having a viscosity of about 1×10⁻³ Pa·s. The ratio of the volumes offluids 528 and 530 was about 1:1. The amplification reagent continuedthrough the downstream storage channel, through tube 544, throughmeasurement zones 510, and then to waste containment region 512. After aset amount of time (12 seconds), the analyzer reopened vent valve 536such that air flows through vent valve 536 (instead of the vent ports).This left some reagent behind in the upstream storage channels 518 and520 on the device. This also results in a single plug of mixedamplification reagent. The 12 seconds of vent-valve closure results inan amplification plug of approximately 50 μL. (Instead of simple timing,another way to trigger the re-opening of the vent valve would be todetect the amplification reagent as it first enters the measurementzones.)

Because the mixed amplification reagent is stable for only a few minutes(usually less than 10 minutes), the mixing was performed less than aminute before use in measurement zone 510. The amplification reagent isa clear liquid, so when it enters the measurement zones, optical densityis at its lowest. As the amplification reagent passed across themeasurement zones, silver was deposited on the captured gold particlesto increase the size of the colloids to amplify the signal. (As notedabove, gold particles were present in the low and high positive controlmeasurement zones and, to the extent that PSA was present in the sample,in the test measurement zone.) Silver can then be deposited on top ofthe already deposited silver, leaving more and more silver deposited inthe measurement zones. Eventually the deposited silver reduces thetransmission of light through the measurement zones. The reduction intransmitted light is proportional to the amount of silver deposited andcan be related to the amount of gold colloids captured on the channelwalls. In a measurement zone where no silver is deposited (the negativecontrol for example, or the test area when the sample contains none ofthe target protein, such as PSA), there will be no (or minimal) increasein optical density. In a measurement zone with significant silverdeposition, the slope and ultimate level of the pattern of increasingoptical density will be high. The analyzer monitors the pattern of thisoptical density during amplification in the test area to determine theconcentration of analyte in the sample. In one version of the test, thepattern is monitored within the first three minutes of amplification.The optical density in each of the measurement zones as a function oftime was recorded and are shown as curves 640, 644, 642, and 646 in FIG.10. These curves corresponded to signals that were produced inmeasurement zones 510-A, 510-B, 510-C, and 510-D, respectively.

After three minutes of amplification, the analyzer stops the test. Nomore optical measurements are recorded and the manifold is disengagedfrom the device. The test result is displayed on the analyzer screen andcommunicated to a printer, computer, or whatever output the user hasselected. The user may remove the device from the analyzer and throw itaway. The sample and all the reagents used in the assay remain in thedevice. The analyzer is ready for another test.

It should be noted that the control of the flow rates of the fluidswithin channel 516 and the measurement zone 510 were important whenflowing fluids through the system. Due to the measurement zone'srelatively small cross sectional area, it served as a bottleneck,controlling the overall flow rate in the system. When the measurementzone contained liquids, the linear flow rates of the fluids in channel516 was about 0.5 mm s⁻¹. Fluids flowing from branching channels 518 and520 into main channel 516 might not have mixed reproducibly at thisrate, as one fluid might have flowed faster than the other, causingunequal portions of fluids 528 and 530 to be mixed. On the other hand,when the measurement zone contained air, the linear flow rates of thefluids in channel 516 and branching channels 518 and 520 were about 15mm s⁻¹. At this higher flow rate, the flow rate in branching channels518 and 520 were equal and reproducible (when vent valve 536 wasclosed), producing reproducible mixing. For this reason, the valveconnected to port 536 was not closed until fluid 542 passed through themeasurement zone to the waste containment region. As noted above,determination of when fluid 542 had exited the measurement zone 510 wasperformed using an optical detector so as to measure transmission oflight through part of measurement zone 510 in combination with afeedback system.

The microfluidic system shown in FIG. 22 was designed such that thevolume of the channel between vent valve 536 and measurement zone 510was larger than the expected volume of the mixed activated silversolution (i.e., the combined portion of fluids 528 and 530 whichtraveled into channel 516 while vent valve 536 was closed). This ensuredthat substantially all of the mixing took place at a relatively highlinear flow rate (since no liquid, and only air, was present in themeasurement zone 510 at this time), and before the activated solutionreached the measurement zone. This configuration helped promotereproducible and equal mixing. For the assay described in this example,it was important to sustain a flow of the activated silver mixturewithin the measurement zone for a few minutes (e.g., 2 to 10 minutes).

This example shows that analysis of a sample in a microfluidic system ofa cassette can be performed by using an analyzer that controls fluidflow in the cassette, and by using feedback from one or more measuredsignals to modulate fluid flow.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed is:
 1. A method of analyzing a microfluidic sample, themethod comprising the steps of: providing a microfluidic sample analyzercomprising a housing with an opening therein, wherein a cassette iscontained in the opening in the housing and, wherein the cassette or acomponent of the cassette includes at least one channel with a fluidsample therein; identifying information about the cassette with anidentification reader positioned on or within the housing; processinginformation input by a user into a user interface positioned within thehousing of the sample analyzer; pressurizing the at least one channel inthe cassette with a pressure-control system positioned within thehousing to move the sample through the at least one channel; activatingan optical system that passes light from a first light sourcepositioning within the housing through a first measurement zone of thecassette; detecting the amount of light transmission through the firstmeasurement zone of the cassette with a first detector of the opticalsystem positioned within the housing opposite the first light source;and analyzing the sample in the cassette with a control systempositioned within the housing which communicates with the identificationreader, the user interface, the pressure-control system, and the opticalsystem.
 2. A method as in claim 1, comprising heating the cassette witha temperature regulating system positioned within the housing of thesample analyzer.
 3. A method as in claim 1, wherein a fluid connectorfluidically interconnects first and second unconnected channels of thecassette so as to cause fluid communication between the first and secondchannels.
 4. A method as in claim 3, wherein, prior to first use of thecassette, at least one of the first and second channels contains astored reagent, the cassette being sealed prior to first use so as tostore the reagent in the cassette for at least one day.
 5. A method asin claim 4, wherein the stored reagent is a liquid.
 6. A method as inclaim 4, wherein, prior to first use of the cassette, at least one ofthe first and second channels contains at least a first and a secondfluid reagent which are separated by a third fluid substantiallyimmiscible with both the first and second fluids.
 7. A method as inclaim 4, wherein, prior to first use of the cassette, a fluid sample iscontained within the fluid connector.
 8. A method as in claim 1, whereinthe identifying information comprises identifying at least one of lotnumber, calibration information, and expiration date of the cassette. 9.A method as in claim 1, wherein the first measurement zone of thecassette includes a meandering channel including a plurality ofsegments, and wherein the first optical system is positioned adjacentmore than one segments of the meandering channel.
 10. A method as inclaim 9, wherein detecting comprises measuring a single signal throughthe more than one segments of the meandering region.
 11. A method as inclaim 1, wherein the cassette includes a plurality of measurement zonesfluidically connected in series, each measurement zone aligned with anoptical system and a light source positioned within the housing, themethod comprising flowing the fluid sample across each of the pluralityof measurement zones and measuring light transmission through each ofthe plurality of measurement zones.
 12. A method as in claim 1,comprising, during essentially the entire analysis, applying asubstantially constant non-zero pressure drop between an inlet to thefirst measurement zone of the cassette and an outlet positioneddownstream of the first measurement zone.
 13. A method as in claim 1,wherein the analyzer comprises a plurality of light sources including atleast a first light source and a second light source adjacent the firstlight source, wherein the first light source is configured to pass lightthrough a first measurement zone of the cassette and the second lightsource is configured to pass light through a second region of thecassette adjacent the first measurement zone, the method comprisingactivating the first light source when the second light source is notactivated, and not activating the second light source unless the firstlight source is deactivated.
 14. A method as in claim 13, comprisingactivating the plurality of light sources sequentially with only onelight source activated at a time.
 15. A method as in claim 1, comprisingaccumulating an opaque material on a portion of a surface of a channelwithin the first measurement zone of the cassette and measuring lighttransmission through the opaque material.
 16. A method as in claim 15,wherein the opaque material comprises a metal.
 17. A method as in claim16, wherein the metal comprises silver.
 18. A method as in claim 15,wherein the opaque material is formed by electroless deposition.
 19. Amethod as in claim 15, wherein the opaque material is deposited byelectroless deposition on a metal colloid.
 20. A method as in claim 19,wherein the metal colloid comprises a gold-conjugated antibody.
 21. Amethod as in claim 15, wherein the opaque material is formed by flowinga metal solution through the channel.
 22. A method as in claim 15,comprising quantitatively determining the opacity of the opaquematerial.
 23. A method as in claim 1, comprising absorbing a fluid inthe cassette with an absorbent material contained in a liquidcontainment region in fluid communication with the first measurementzone.
 24. A method as in claim 1, comprising absorbing substantially allliquids flowing in the cassette in a liquid containment region in fluidcommunication with the first measurement zone, while allowing any gasesto escape from an outlet of the cassette.
 25. A method as in claim 1,wherein the fluid sample comprises whole blood.